Secreted salivary zsig32 polypeptides

ABSTRACT

The present invention relates to polynucleotide and polypeptide molecules for secreted salivary zsig32 polypeptides. The polypeptides, and polynucleotides encoding them modulate adhesion or modulate or indicate salivary gland function. The present invention also includes antibodies and binding proteins for the zsig32 polypeptides.

BACKGROUND OF THE INVENTION

[0001] The salivary glands synthesize and secrete a number of proteinshaving diverse biological functions. Such proteins facilitatelubrication of the oral cavity (e.g., mucins and proline-rich proteins),remineralization (e.g., statherin and ionic proline-rich proteins) anddigestion (e.g., amylase, lipase and proteases) and provideanti-microbial (e.g., proline-rich proteins, lysozyme, histatins andlactoperoxidase) and mucosal integrity maintenance (e.g., mucins)capabilities. In addition, saliva is a rich source of growth factorssynthesized by the salivary glands. For example, saliva is known tocontain epidermal growth factor (EGF), nerve growth factor (NGF),transforming growth factor-alpha (TGF-α), transforming growthfactor-beta (TGF-β), insulin, insulin-like growth factors I and II(IGF-I and IGF-II) and fibroblast growth factor (FGF). See, for example,Zelles et al., J. Dental. Res. 74(12): 1826-32, 1995. Synthesis ofgrowth factors by the salivary gland is believed to beandrogen-dependent and to be necessary for the health of the oral cavityand gastrointestinal tract.

[0002] Some salivary gland-produced proteins, such as EGF, are believedto have systemic wound healing effects. Effective wound healing appearsto require extended exposure of afflicted tissue to growth factors,which may be facilitated in the oral cavity and gastrointestinal tractby mucin at the epithelial/environmental interface acting to capturesaliva growth factors. Also, combinations of growth factors, such asthose found in saliva, may be necessary for optimal wound healing.Moreover, protease inhibitors, which are also produced by the salivaryglands, appear to facilitate growth factor activity.

[0003] In addition, saliva contains adhesive proteins having protectiveproperties with regard to infection by exogenous microorganisms. Suchadhesive proteins bind exogenous microorganisms and facilitate thedegradation or expulsion thereof. From this rich source of biologicallyrelevant proteins, new secreted proteins are sought. Also, given theimportance and variety of saliva proteins, conditions involvinginadequate saliva production or secretion have inspired investigativeeffort.

[0004]Rattus norvegicus common salivary protein 1 (U00964_(—)1) and amurine homolog of common salivary protein 1 (S76879_(—)1) have beendiscovered and characterized. See, for example, Girard et al., J. Biol.Chem. 268(35): 26592-601, 1993. Common salivary protein 1 is sodesignated as a result of the expression thereof in cells of all majorsalivary glands. Evidence exists that such expression isandrogen-regulated in the rat submandibular gland. Common salivaryprotein 1 does not include structural features associated with manyother salivary proteins, including tandemly repeated sequences, a highdensity of charged residues and/or an unusually large proportion of afew amino acids. Common salivary protein 1 is somewhat homologous tospermine binding proteins discussed below, but lacks the highly acidiccarboxy terminal domain thereof. Thus, the proteins may beevolutionarily related without being functionally related.

[0005] Salivary glands share significant features with other glands,such as the prostate gland. For example, the salivary glands andprostate gland are classified as slow replicators with respect to theirproliferative capacity. See, for example, Zajicek, Med. Hypotheses7(10): 1241-51, 1981. Such slow replicators exhibit similar onotgeniesand proceed during regeneration and neoplasia through similar stages.The prostate gland also appears to produce growth factors, such as EGFand NGF, and other biologically important proteins, such as kallikreins.See, for example, Hiramatsu et al., Biochem. Int. 17 (2): 311-7, 1988,Harper et al., J. Biol. Chem. 257(14): 8541-8, 1982 and Brady et al.,Biochemistry 28(12): 5203-10, 1988. Prostate gland function also appearsto be androgen-dependent. Consequently, proteins associated with theprostate gland are also sought.

[0006] Glandular function is believed to be androgen-dependent.Expression of secreted glycoproteins, having spermine-binding activity,by mouse and rat prostate has also been postulated to beandrogen-dependent. See, for example, Mills et al., Nucleic Acids Res.15: 7709-24, 1987. Spermine-binding protein mRNA expression appears tobe induced by exposure to androgens, with an increase therein by 2-3fold being observed within 16 hours and continuing for several days.Thus, intracellular levels of specific hormone-dependent mRNA, such asspermine binding protein mRNA, are useful markers of hormone action.See, Labrie et al., Endocrinology 124 (6): 2745-54, 1989.Spermine-binding protein is also useful for studying cAMP-independentprotein kinases, because the protein is under androgenic control throughthe action of such kinases. See, for example, Goueli et al., Biochem. J.(England) 230(2): 293-302, 1985.

[0007] Spermine-binding proteins bind to polyamines, such as spermine.Prostatic fluid, for example, is rich in polyamines, andspermine-binding proteins have therefore been postulated to serve ascarriers of such polyamines in the seminal fluid. Spermine-bindingproteins may also be useful to disrupt spermine-mediated pig lenssoluble protein aggregation to form cataracts. See, for example,Maekawa, Mie. Med. J. 39(2): 221-8, 1989. In addition, spermine binds toand is believed to modulate the activity of the N-methyl-D-aspartate(NMDA) receptor, a ligand-gated ion channel (Bergeron et al., J. Med.Chem. 38: 425-8, 1995) Also, spermine is believed to effect vascularsmooth muscle cell contractility via inhibition of myosin phosphatase(Sward et al., Am. J. Physiology 269(3 pt. 1): C563-71, 1995). Thus,homologs of spermine-binding proteins are sought.

[0008] The present invention provides such polypeptides for these andother uses that should be apparent to those skilled in the art from theteachings herein.

SUMMARY OF THE INVENTION

[0009] Within one aspect the invention provides an isolated polypeptidecomprising a sequence of amino acid residues that is at least 80%identical in amino acid sequence to residues 23-178 of SEQ ID NO:2.Within one embodiment the polypeptide is at least 90% identical in aminoacid sequence to residues 23-178 of SEQ ID NO:2. Within anotherembodiment the polypeptide comprises residues 7-178 of SEQ ID NO:2.Within yet another embodiment the polypeptide comprises residues 1-178of SEQ ID NO:2. Within still another embodiment the polypeptide is atleast 1 kb in length. Within yet another embodiment the polypeptide iscovalently linked to a moiety selected from the group consisting ofaffinity tags, toxins, radionucleotides, enzymes and fluorophores.Within a related embodiment the moiety is an affinity tag selected fromthe group consisting of polyhistidine, FLAG, Glu-Glu, glutathione Stransferase and an immunoglobulin heavy chain constant region. Withinanother related embodiment the polypeptide further comprises aproteolytic cleavage site between the sequence of amino acid residuesand the affinity tag.

[0010] Within another aspect is provided an expression vector comprisingthe following operably linked elements: a transcription promoter; a DNAsegment encoding a polypeptide as described above; and a transcriptionalterminator. Within a related embodiment the DNA segment encodes apolypeptide covalently linked to an affinity tag selected from the groupconsisting of polyhistidine, FLAG, Glu-Glu, glutathione S transferaseand an immunoglobulin heavy chain constant region. Within a relatedembodiment the DNA further encodes a secretory signal sequence operablylinked to said polypeptide. Within another embodiment the secretorysignal sequence encodes residues 7-22 of SEQ ID NO:2. Within a relatedembodiment the secretory signal sequence encodes residues 1-22 of SEQ IDNO:2. Within another related embodiment is provided a cultured cell intowhich has been introduced an expression vector as described above,wherein the cell expresses the polypeptide encoded by the DNA segment.

[0011] Within another aspect is provided a method of producing a proteincomprising: culturing a cell into which has been introduced anexpression vector as described above, whereby the cell expresses theprotein encoded by the DNA segment; and recovering the expressedprotein.

[0012] Within another aspect is provided a pharmaceutical compositioncomprising a polypeptide as described above in combination with apharmaceutically acceptable vehicle.

[0013] Within other aspects are provided an antibody that specificallybinds to an epitope of a polypeptide as described above and a bindingprotein that specifically binds to an epitope of a polypeptide asdescribed above.

[0014] Within a further aspect is provided an isolated polynucleotideencoding a polypeptide as described above. Within one embodiment isprovided an isolated polynucleotide as described above wherein thepolynucleotide is selected from the group consisting of, a) a sequenceof nucleotides from nucleotide 168 to nucleotide 704 of SEQ ID NO:1; b)a sequence of nucleotides from nucleotide 186 to nucleotide 704 of SEQID NO:2; c) a sequence of nucleotides from nucleotide 234 to nucleotide704 of SEQ ID NO:2; d) a sequence of nucleotides from nucleotide 246 tonucleotide 704 of SEQ ID NO:2; e) allelic variants of a), b), c) or d);and f) nucleotide sequences complementary to a), b), c), d) or e).Within another embodiment the polynucleotide is from 471 to 853nucleotides in length. Within another embodiment the polynucleotidecomprises nucleotide 1 to nucleotide 534 of SEQ ID NO:20. Within anotherembodiment the polynucleotide is DNA.

[0015] Within another aspect of the invention is provided anoligonucleotide probe or primer comprising 14 contiguous nucleotides ofa polynucleotide of SEQ ID NO:20 or a sequence complementary to SEQ IDNO:20.

[0016] Within yet another aspect is provided a method for detecting agenetic abnormality in a patient, comprising: obtaining a genetic samplefrom a patient; incubating the genetic sample with a polynucleotidecomprising at least 14 contiguous nucleotides of SEQ ID NO:1 or thecomplement of SEQ ID NO:1, under conditions wherein said polynucleotidewill hybridize to complementary polynucleotide sequence, to produce afirst reaction product; comparing said first reaction product to acontrol reaction product, wherein a difference between said firstreaction product and said control reaction product is indicative of agenetic abnormality in the patient.

[0017] Within a further aspect is provided a DNA construct encoding apolypeptide fusion, said fusion comprising a secretory signal sequenceselected from the group consisting of: (a) amino acid residues 1-22 ofSEQ ID NO:2; and (b) amino acid residues 7-22 of SEQ ID NO:2; whereinthe secretory signal sequence is operably linked to an additionalpolypeptide.

[0018] Within another aspect is provided a method for detecting zsig32polypeptides comprising: exposing a polypeptide containing sample to anantibody attached to a solid support, wherein said antibody binds to anepitope of a zsig32 polypeptide; washing said immobilizedantibody-polypeptide to remove unbound contaminants; exposing theimmobilized antibody-polypeptide to a second antibody directed to asecond epitope of a zsig32 polypeptide, wherein the second antibody isassociated with a detectable label; and detecting the detectable label.

[0019] These and other aspects of the invention will become evident uponreference to the following detailed description of the invention and theattached drawing.

BRIEF DESCRIPTION OF THE DRAWING

[0020] The FIGURE illustrates an alignment of mouse ventral prostatespermine-binding protein (SPBP_MOUSE), Rattus norvegicus common salivaryprotein 1 (U00964_(—)1), a murine common salivary protein 1(S76879_(—)1) and a zsig32 polypeptide of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0021] Prior to setting forth the invention in detail, it may be helpfulto the understanding thereof to define the following terms:

[0022] The term “affinity tag” is used herein to denote a polypeptidesegment that can be attached to a second polypeptide to provide forpurification or detection of the second polypeptide or provide sites forattachment of the second polypeptide to a substrate. In principal, anypeptide or protein for which an antibody or other specific binding agentis available can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

[0023] The term “allelic variant” denotes any of two or more alternativeforms of a gene occupying the same chromosomal locus. Allelic variationarises naturally through mutation, and may result in phenotypicpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequence. The term allelic variant is also used herein todenote a protein encoded by an allelic variant of a gene.

[0024] The terms “amino-terminal” and “carboxyl-terminal” are usedherein to denote positions within polypeptides and proteins. Where thecontext allows, these terms are used with reference to a particularsequence or portion of a polypeptide or protein to denote proximity orrelative position. For example, a certain sequence positionedcarboxyl-terminal to a reference sequence within a protein is locatedproximal to the carboxyl terminus of the reference sequence, but is notnecessarily at the carboxyl terminus of the complete protein.

[0025] The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

[0026] The term “complements of polynucleotide molecules” denotespolynucleotide molecules having a complementary base sequence andreverse orientation as compared to a reference sequence. For example,the sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

[0027] The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

[0028] The term “expression vector” denotes a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestoperably linked to additional segments that provide for itstranscription. Such additional segments may include promoter andterminator sequences, and may optionally include one or more origins ofreplication, one or more selectable markers, an enhancer, apolyadenylation signal, and the like. Expression vectors are generallyderived from plasmid or viral DNA, or may contain elements of both.

[0029] The term “isolated”, when applied to a polynucleotide molecule,denotes that the polynucleotide has been removed from its naturalgenetic milieu and is thus free of other extraneous or unwanted codingsequences, and is in a form suitable for use within geneticallyengineered protein production systems. Such isolated molecules are thosethat are separated from their natural environment and include cDNA andgenomic clones. Isolated DNA molecules of the present invention are freeof other genes with which they are ordinarily associated, but mayinclude naturally occurring 5′ and 3′ untranslated regions such aspromoters and terminators. The identification of associated regions willbe evident to one of ordinary skill in the art (see for example, Dynanand Tijan, Nature 316:774-78, 1985). When applied to a protein, the term“isolated” indicates that the protein is found in a condition other thanits native environment, such as apart from blood and animal tissue. In apreferred form, the isolated protein is substantially free of otherproteins, particularly other proteins of animal origin. It is preferredto provide the protein in a highly purified form, i.e., greater than 95%pure, more preferably greater than 99% pure.

[0030] The term “operably linked”, when referring to DNA segments,denotes that the segments are arranged so that they function in concertfor their intended purposes, e.g. transcription initiates in thepromoter and proceeds through the coding segment to the terminator.

[0031] The term “ortholog” denotes a polypeptide or protein obtainedfrom one species that is the functional counterpart of a polypeptide orprotein from a different species. Sequence differences among orthologsare the result of speciation.

[0032] “Paralogs” are distinct but structurally related proteins made byan organism. Paralogs are believed to arise through gene duplication.For example, α-globin, β-globin, and myoglobin are paralogs of eachother.

[0033] The term “polynucleotide” denotes a single- or double-strandedpolymer of deoxyribonucleotide or ribonucleotide bases read from the 5′to the 3′ end. Polynucleotides include RNA and DNA, and may be isolatedfrom natural sources, synthesized in vitro, or prepared from acombination of natural and synthetic molecules. Sizes of polynucleotidesare expressed as base pairs (abbreviated “bp”), nucleotides (“nt”), orkilobases (“kb”). Where the context allows, the latter two terms maydescribe polynucleotides that are single-stranded or double-stranded.When the term is applied to double-stranded molecules it is used todenote overall length and will be understood to be equivalent to theterm “base pairs”. It will be recognized by those skilled in the artthat the two strands of a double-stranded polynucleotide may differslightly in length and that the ends thereof may be staggered as aresult of enzymatic cleavage; thus all nucleotides within adouble-stranded polynucleotide molecule may not be paired. Such unpairedends will in general not exceed 20 nt in length.

[0034] “Probes and/or primers” as used herein can be RNA or DNA. DNA canbe either cDNA or genomic DNA. Polynucleotide probes and primers aresingle or double-stranded DNA or RNA, generally syntheticoligonucleotides, but may be generated from cloned cDNA or genomicsequences or its complements. Analytical probes will generally be atleast 20 nucleotides in length, although somewhat shorter probes (14-17nucleotides) can be used. PCR primers are at least 5 nucleotides inlength, preferably 15 or more nt, more preferably 20-30 nt. Shortpolynucleotides can be used when a small region of the gene is targetedfor analysis. For gross analysis of genes, a polynucleotide probe maycomprise an entire exon or more. Probes can be labeled to provide adetectable signal, such as with an enzyme, biotin, a radionuclide,fluorophore, chemiluminescer, paramagnetic particle and the like, whichare commercially available from many sources, such as Molecular Probes,Inc., Eugene, Oreg., and Amersham Corp., Arlington Heights, Ill., usingtechniques that are well known in the art.

[0035] The term “promoter” denotes a portion of a gene containing DNAsequences that provide for the binding of RNA polymerase and initiationof transcription. Promoter sequences are commonly, but not always, foundin the 5′ non-coding regions of genes.

[0036] The term “receptor” denotes a cell-associated protein that bindsto a bioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-domain structure comprising an extracellular ligand-binding domainand an intracellular effector domain that is typically involved insignal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. Most nuclear receptors also exhibit amulti-domain structure, including an amino-terminal, transactivatingdomain, a DNA binding domain and a ligand binding domain. In general,receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g.,thyroid stimulating hormone receptor, beta-adrenergic receptor) ormultimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor,GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6receptor).

[0037] The term “secretory signal sequence” denotes a DNA sequence thatencodes a polypeptide (a “secretory peptide”) that, as a component of alarger polypeptide, directs the larger polypeptide through a secretorypathway of a cell in which it is synthesized. The larger peptide iscommonly cleaved to remove the secretory peptide during transit throughthe secretory pathway.

[0038] The term “splice variant” is used herein to denote alternativeforms of RNA transcribed from a gene. Splice variation arises naturallythrough use of alternative splicing sites within a transcribed RNAmolecule, or less commonly between separately transcribed RNA molecules,and may result in several mRNAs transcribed from the same gene. Splicevariants may encode polypeptides having altered amino acid sequence. Theterm splice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

[0039] Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

[0040] All references cited herein are incorporated by reference intheir entirety.

[0041] The present invention is based in part upon the discovery of anovel DNA sequence that encodes a polypeptide characterized by homologyto mouse ventral prostate spermine-binding protein (SEQ ID NO: 3). See,for example, Mills et al., Nucleic Acids Res. 15: 7709-24, 1987.Possibly more significant is the homology of zsig32 polypeptide toRattus norvegicus common salivary protein (U00964_(—)1; SEQ ID NO: 4),as discussed in Girard et al., J. Biol. Chem., 268(35): 26592-601, 1993,and murine common salivary protein 1 (S76879_(—)1; SEQ ID NO: 5).

[0042] In addition, the zsig32 polypeptides of the present inventionpreferably incorporate one potential N-glycosylation site at amino acid167 (Asn) of SEQ ID NO: 2. Zsig32 polypeptides of the present inventionalso preferably incorporate two potential tyrosine sulfatation sites atamino acids 40 and 155 of SEQ ID NO: 2. Preferably, one potentialprotein kinase C phosphorylation site is located at amino acid 107 (Tyr)of SEQ ID NO: 2. Such putative sites of phosphorylation may indicatethat zsig32, like spermine-binding protein, is regulated bycAMP-independent protein kinases. See, for example, Goueli et al.,Biochem. J. (England) 230(2): 293-302, 1985. In addition, the zsig32polypeptides of the present invention preferably incorporate threepotential casein kinase II phosphorylation sites at amino acids 35(Ser), 36 (Tyr) and 107 (Tyr) of SEQ ID NO: 2. Such sites may indicatethat zsig32 may be regulated by casein kinase II. Preferably, threepotential N-myristoylation sites are located at amino acids 46, 72 and120 of SEQ ID NO: 2). These sites are not 100% conserved in the alignedcommon salivary proteins.

[0043] Zsig32 polypeptides also preferably incorporate an adhesion motif(amino acid residues 63-65 of SEQ ID NO: 2). According tothree-dimensional analysis of polypeptide structure, the adhesion motifappears to be exposed. Thus, zsig32 polypeptides and fragments thereof,incorporating such an adhesion motif, appear to be useful in the studyof adhesion, as is more fully described herein. Also useful in thisregard are fusion proteins containing zsig32 polypeptide or an exposedadhesion motif-containing fragment thereof.

[0044] The novel polypeptides of the present invention, designatedzsig32 polypeptides, were initially identified by querying an ESTdatabase for secretory signal sequences characterized by an upstreammethionine start site, a hydrophobic region of approximately 13 aminoacids and a cleavage site (SEQ ID NO: 6, wherein cleavage occurs betweenthe alanine and glycine amino acid residues) in an effort to select forsecreted proteins. Polypeptides corresponding to ESTs meeting thosesearch criteria were compared to known sequences to identify secretedproteins having homology to known ligands. A single EST sequence wasdiscovered and predicted to be related to a secreted spermine-bindingprotein found in rat ventral prostate (SPBP). See, for example, Mills etal., Nucleic Acids Res. 15: 7709-24, 1987. Homology was also discoveredbetween zsig32 polypeptide and Rattus norvegicus common salivary protein1 (U00964_(—)1), as discussed in Girard et al., J. Biol. Chem., 268(35):26592-601, 1993, and a murine common salivary protein 1 (S76879_(—)1).

[0045] The full sequence of the zsig32 polypeptide was obtained from asingle clone believed to contain it, wherein the clone was obtained froma parotid gland tissue library. Other libraries that might also besearched for such clones include submandibular gland, salivary gland,prostate, trachea, colon, stomach, prostate tumor, lung, thyroid, tonguetumor and the like.

[0046] Analysis of the tissue distribution of the mRNA corresponding tothis novel DNA by Northern blot analysis using a synthetic probe (SEQ IDNO: 7) showed that expression was highest in trachea, lower in prostate,and apparent but further decreased in colon and stomach. A tissuedistribution analysis by Dot blot showed extremely high expression insalivary gland, with significantly less expression in trachea andapparent but further decreased expression in prostate. In evaluatingthese results it is important to note that the Northern blot analysisdid not include the salivary gland. A single transcript size ofapproximately 1 kb was observed.

[0047] The nucleotide sequence of the N-terminal EST is described in SEQID NO: 1, and its deduced amino acid sequence is described in SEQ ID NO:2. Analysis of the DNA encoding a zsig32 polypeptide (SEQ ID NO: 1)revealed an open reading frame encoding either 178 (Met at amino acidnumber 1 of SEQ ID NO: 2 is the start site) or 172 amino acids (Met atamino acid number 7 of SEQ ID NO: 2 is the start site). The open readingframe comprises a signal peptide of either 22 amino acid residues(residue 1 to residue 22 of SEQ ID NO: 2; Met at position 1 is the startsite) or 16 amino acid residues (residue 7 to residue 22 of SEQ ID NO:2; Met at position 7 is the start site) and a mature polypeptide of 156amino acids (residue 23 to residue 178 of SEQ ID NO: 2). Those skilledin the art will recognize that predicted secretory signal sequencedomain boundaries are approximations based on primary sequence content,and may vary slightly; however, such estimates are generally accurate towithin ±4 amino acid residues. Therefore the present invention alsoincludes the polypeptides having amino acid sequences comprising aminoacid residues 20-178 of SEQ ID NO:2, residues 21-178 of SEQ ID NO:2,residues 22-178 of SEQ ID NO:2, residues 23-178 of SEQ ID NO:2, residues24-178 of SEQ ID NO:2, residues 25-178, residues 26-178 of SEQ ID NO:2and residues 27-178 of SEQ ID NO:2 as well as the polynucleotidesencoding them.

[0048] An alignment was prepared including zsig32 polypeptide, mouseventral prostate spermine-binding protein SPBP_MOUSE (SEQ ID NO: 3; Metat position 7) rat common salivary protein 1 (U00964_(—)1; SEQ ID NO: 4;Met at positions 1 and 7), and murine common salivary protein 1(S76879_(—)1; SEQ ID NO: 5; Met at positions 1 and 7). That alignment,as shown in the FIGURE, revealed a block of high percent identity in thesignal sequence and a block of significant percent identity in themature protein corresponding to the region of SEQ ID NO: 2 from aminoacid residue 7 (Met), corresponding to aligned residue 7, to amino acidresidue 217 (Glu), corresponding to aligned residue 167. When compared,the most conserved sequences are in the signal sequence. Also, the twocommon salivary proteins and zsig32 share a six amino acid presequence(residues 1-6 of SEQ ID NOS. 4, 5 and 6). In addition, the C-terminaltail of SPBP_MOUSE appears to be longer than that of zsig32 and to becharacterized by high asparagine/aspartic acid concentration. TheC-terminal tail of SPBP_MOUSE is believed to participate in sperminebinding. In contrast, the C-terminal tails of the salivary proteins areshorter than that of zsig32. Neither zsig32 nor the salivary proteinsincorporate a region of high asparagine/aspartic acid concentration,potentially indicating alternative regulation or specificity.

[0049] Within the region of significant identity in the maturepolypeptides, the following percent identity figures are observed forthe deduced amino acid sequence of zsig32 polypeptide (SEQ ID NO: 2),SPEP_MOUSE (SEQ ID NO: 3), rat common salivary protein 1, U00964_(—)1(SEQ ID NO: 4), and murine common salivary protein 1, S76879_(—)1 (SEQID NO: 5). Zsig32 U00964_1 S76879_1 SPEP_MOUSE Zsig32 100 31 29 29U00964_1 31 100 44 29 S76879_1 29 44 100 28 SPEP_MOUSE 29 29 28 100

[0050] Highly conserved amino acids can be used as a tool to identifyzsig32 polypeptides or other proteins characterized by salivary glandfunction indication or modulation, spermine or polyamine bindingcapability, cAMP-independent protein kinase- or androgen-regulationsusceptibility or the like. For instance, reversetranscription-polymerase chain reaction (RT-PCR) can be used to amplifysequences encoding a conserved motif from RNA obtained from a variety oftissue sources. In particular, the following primers are useful for thispurpose.

[0051] 1) Amino acids 14-19 of SEQ ID NO: 2 (corresponding tonucleotides 40-57 of SEQ ID NO: 1, nucleotides 40-57 of SEQ ID NO:20 andtheir complements);

[0052] 2) Amino acids 31-36 of SEQ ID NO: 2 (corresponding tonucleotides 91-108 of SEQ ID NO: 1, nucleotides 97-108 of SEQ ID NO:20and their complements);

[0053] 3) Amino acids 154-159 of SEQ ID NO: 2 (corresponding tonucleotides 460-477 of SEQ ID NO: 1, nucleotides 460-477 of SEQ ID NO:20and their complements); and

[0054] 4) Amino acids 109-114 of SEQ ID NO: 2 (corresponding tonucleotides 325-342 of SEQ ID NO: 1, nucleotides 325-342 of SEQ IDNO:20, and their complements).

[0055] The activity of polypeptides identified by such robes or ofpolypeptides encoded by polynucleotides identified by such probes can bedetermined by methods that are known in the art as generally describedherein.

[0056] Oligonucleotide probes based on the polynucleotide sequence ofSEQ ID NO:1 can be used to localize the zsig32 gene to a particularchromosome. Radiation hybrid mapping is a somatic cell genetic techniquedeveloped for constructing high-resolution, contiguous maps of mammalianchromosomes (Cox et al., Science 250:245-50, 1990). Partial or fullknowledge of a gene's sequence allows one to design PCR primers suitablefor use with chromosomal radiation hybrid mapping panels. Radiationhybrid mapping panels are commercially available which cover the entirehuman genome, such as the Stanford G3 RH Panel and the GeneBridge 4 RHPanel (Research Genetics, Inc., Huntsville, Ala.). These panels enablerapid, PCR-based chromosomal localizations and ordering of genes,sequence-tagged sites (STSs), and other nonpolymorphic and polymorphicmarkers within a region of interest. This includes establishing directlyproportional physical distances between newly discovered genes ofinterest and previously mapped markers. The precise knowledge of agene's position can be useful for a number of purposes, including: 1)determining if a sequence is part of an existing contig and obtainingadditional surrounding genetic sequences in various forms, such as YACs,BACs or cDNA clones; 2) providing a possible candidate gene for aninheritable disease which shows linkage to the same chromosomal region;and 3) cross-referencing model organisms, such as mouse, which may aidin determining what function a particular gene might have.

[0057] Sequence tagged sites (STSs) can also be used independently forchromosomal localization. An STS is a DNA sequence that is unique in thehuman genome and can be used as a reference point for a particularchromosome or region of a chromosome. An STS is defined by a pair ofoligonucleotide primers that are used in a polymerase chain reaction tospecifically detect this site in the presence of all other genomicsequences. Since STSs are based solely on DNA sequence they can becompletely described within an electronic database, for example,Database of Sequence Tagged Sites (dbSTS), GenBank, (National Center forBiological Information, National Institutes of Health, Bethesda, Md.http://www.ncbi.nlm.nih.gov), and can be searched with a gene sequenceof interest for the mapping data contained within these short genomiclandmark STS sequences.

[0058] The results of chromosome mapping experiments, as more fullydescribed in Example 3 hereof, showed that the zsig32 gene maps 7.47 cRfrom the top of the human chromosome 16 linkage group on the WICGRradiation hybrid map. Relative to the centromere, its nearest proximalmarker was WI-7742 and its nearest distal maker was WI-3061. The use ofsurrounding markers positioned the zsig32 gene in the 16p13.3 region onthe integrated LDB chromosome 16 map.

[0059] The present invention also provides polynucleotide molecules,including DNA and RNA molecules, that encode the zsig32 polypeptidesdisclosed herein. Those skilled in the art will readily recognize that,in view of the degeneracy of the genetic code, considerable sequencevariation is possible among these polynucleotide molecules. SEQ ID NO:20is a degenerate DNA sequence that encompasses all DNAs that encode thezsig32 polypeptide of SEQ ID NO:2. Those skilled in the art willrecognize that the degenerate sequence of SEQ ID NO:20 also provides allRNA sequences encoding SEQ ID NO:2 by substituting U for T. Thus, zsig32polypeptide-encoding polynucleotides comprising nucleotide 61 tonucleotide 534 of SEQ ID NO:20, nucleotide 22 to nucleotide 534 of SEQID NO:20 and nucleotide 1 to nucleotide 534 of SEQ ID NO:20 and theirRNA equivalents are contemplated by the present invention. Table 1 setsforth the one-letter codes used within SEQ ID NO:20 to denote degeneratenucleotide positions. “Resolutions” are the nucleotides denoted by acode letter. “Complement” indicates the code for the complementarynucleotide(s). For example, the code Y denotes either C or T, and itscomplement R denotes A or G, A being complementary to T, and G beingcomplementary to C. TABLE 1 Nucleotide Resolution Nucleotide ComplementA A T T C C G G G G C C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T KG|T M A|C S C|G S C|G W A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G VA|C|G B C|G|T D A|G|T H A|C|T N A|C|G|T N A|C|G|T

[0060] The degenerate codons used in SEQ ID NO:20, encompassing allpossible codons for a given amino acid, are set forth in Table 2. TABLE2 One Amino Letter Degenerate Acid Code Codons Codon Cys C TGC TGT TGYSer S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro P CCACCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGN AsnN AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CAR HisH CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AAR Met MATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTN Val VGTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGG TGGTer . TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN

[0061] One of ordinary skill in the art will appreciate that someambiguity is introduced in determining a degenerate codon,representative of all possible codons encoding each amino acid. Forexample, the degenerate codon for serine (WSN) can, in somecircumstances, encode arginine (AGR), and the degenerate codon forarginine (MGN) can, in some circumstances, encode serine (AGY). Asimilar relationship exists between codons encoding phenylalanine andleucine. Thus, some polynucleotides encompassed by the degeneratesequence may encode variant amino acid sequences, but one of ordinaryskill in the art can easily identify such variant sequences by referenceto the amino acid sequence of SEQ ID NO:2. Variant sequences can bereadily tested for functionality as described herein.

[0062] One of ordinary skill in the art will also appreciate thatdifferent species can exhibit “preferential codon usage.” In general,see, Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al.Curr. Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene 13:355-64, 1981;Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res.14:3075-87, 1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As usedherein, the term “preferential codon usage” or “preferential codons” isa term of art referring to protein translation codons that are mostfrequently used in cells of a certain species, thus favoring one or afew representatives of the possible codons encoding each amino acid (SeeTable 2). For example, the amino acid threonine (Thr) may be encoded byACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonlyused codon; in other species, for example, insect cells, yeast, virusesor bacteria, different Thr codons may be preferential. Preferentialcodons for a particular species can be introduced into thepolynucleotides of the present invention by a variety of methods knownin the art. Introduction of preferential codon sequences intorecombinant DNA can, for example, enhance production of the protein bymaking protein translation more efficient within a particular cell typeor species. Therefore, the degenerate codon sequence disclosed in SEQ IDNO:20 serves as a template for optimizing expression of polynucleotidesin various cell types and species commonly used in the art and disclosedherein. Sequences containing preferential codons can be tested andoptimized for expression in various species, and tested forfunctionality as disclosed herein.

[0063] Based upon homology to spermine binding protein, zsig32polypeptides may be used in the study of cAMP-independent proteinkinases or androgens. Androgens mediate cAMP-independent protein kinaselevels, which in turn mediate phosphorylation of zsig32 polypeptides. Inan embodiment of the present invention, immobilized zsig32 polypeptidesare incubated with γ-³²P-ATP and a cAMP-independent protein kinase, andincorporation of ³²P into zsig32 polypeptides is monitored by knowntechniques. Alternatively, cells that express or are engineered toexpress zsig32 polypeptides and that endogenously express one or morecAMP-independent protein kinase are incubated with androgens andγ-³²P-ATP. The cells are then lysed and incorporation of ³²P into zsig32polypeptides is monitored by known techniques. In addition, sperminebinding protein mRNA has been suggested as a marker for specificandrogen activity, as described in Labrie et al., Endocrinology 124(6):2745-54, 1989. Zsig32 polypeptide mRNA may also be useful for thatpurpose.

[0064] As shown in the FIGURE and described herein, zsig32 polypeptidesalso exhibit homology to common salivary protein 1 isolated from rat andmouse. Moreover, zsig32 polypeptides are characterized by a limitedtissue distribution. While the Northern blot and Dot blot analyses arenot in complete agreement, a high level of expression of zsig32polypeptide was observed in the salivary gland. Consequently, anotheraspect of the present invention involves the detection of zsig32polypeptides in the serum or tissue biopsy of a patient undergoingevaluation for salivary gland function or dysfunction. Such zsig32polypeptides can be detected using immunoassay techniques and antibodiescapable of recognizing zsig32 polypeptide epitopes.

[0065] More specifically, the present invention contemplates methods fordetecting zsig32 polypeptide comprising:

[0066] exposing a solution possibly containing zsig32 polypeptide to anantibody attached to a solid support, wherein said antibody binds to afirst epitope of a zsig32 polypeptide;

[0067] washing said immobilized antibody-polypeptide to remove unboundcontaminants;

[0068] exposing the immobilized antibody-polypeptide to a secondantibody directed to a second epitope of a zsig32 polypeptide, whereinthe second antibody is associated with a detectable label; and

[0069] detecting the detectable label. Serum or biopsy zsig32polypeptide concentration (relative to normal serum or tissueconcentration) may be indicative of dysfunction of the salivary gland.Salivary gland dysfunctions include digestive dysfunction, wound healingdysfunction, inadequate saliva production or composition, mucosalintegrity breakdown, failure or diminished anti-microbial-function.Detection of zsig32 polypeptide at relatively high levels in the tracheamay indicate that such polypeptides may serve as a marker of lungdysfunction. Examples of conditions associated with salivary gland orlung dysfunction include salivary gland carcinoma, sarcoidosis,pneumocystic carinii (particularly as associated with AIDS patients),emphysema, chronic bronchitis, cystic fibrosis, ARDS, SIDS or the like.In addition, zsig32 polypeptides are expressed in the prostate, albeitat a lower level than in the salivary gland and trachea. The prostategland is androgen regulated and shares other properties with salivaryglands. Consequently, dysfunction thereof, such as prostateadenocarcinoma or the like, may also be detected using zsig32polypeptides.

[0070] Also, the salivary glands synthesize and secrete a number ofproteins having diverse biological functions. Such proteins facilitatelubrication of the oral cavity (e.g., mucins and proline-rich proteins),remineralization (e.g., statherin and ionic proline-rich proteins),digestion (e.g., amylase, lipase and proteases), provide anti-microbial(e.g., proline-rich proteins, lysozyme, histatins and lactoperoxidase)and mucosal integrity maintenance (e.g., mucins) capabilities. Inaddition, saliva is a rich source of growth factors synthesized by thesalivary glands. For example, saliva is known to contain epidermalgrowth factor (EGF), nerve growth factor (NGF), transforming growthfactor-alpha (TGF-α), transforming growth factor-beta (TGF-β), insulin,insulin-like growth factors I and II (IGF-I and IGF-II) and fibroblastgrowth factor (FGF). See, for example, Zelles et al., J. Dental. Res.74: 1826-32, 1995. Synthesis of growth factors by the salivary gland isbelieved to be androgen-dependent and to be necessary for the health ofthe oral cavity and gastrointestinal tract.

[0071] Thus, zsig32 polypeptides, agonists or antagonists thereof may betherapeutically useful for aiding digestion. To verify the presence ofthis capability in zsig32 polypeptides, agonists or antagonists of thepresent invention, such zsig32 polypeptides, agonists or antagonists areevaluated with respect to their ability to break down starch accordingto procedures known in the art. If desired, zsig32 polypeptideperformance in this regard can be compared to digestive enzymes, such asamylase, lipase, proteases and the like. In addition, zsig32polypeptides or agonists or antagonists thereof may be evaluated incombination with one or more digestive enzymes to identify synergisticeffects.

[0072] Also, zsig32 polypeptides, agonists or antagonists thereof may betherapeutically useful for promoting wound healing. To verify thepresence of this capability in zsig32 polypeptides, agonists orantagonists of the present invention, such zsig32 polypeptides, agonistsor antagonists are evaluated with respect to their ability to facilitatewound healing according to procedures known in the art. If desired,zsig32 polypeptide performance in this regard can be compared to growthfactors, such as EGF, NGF, TGF-α, TGF-β, insulin, IGF-I, IGF-II,fibroblast growth factor (FGF) and the like. In addition, zsig32polypeptides or agonists or antagonists thereof may be evaluated incombination with one or more growth factors to identify synergisticeffects.

[0073] In addition, zsig32 polypeptides, agonists or antagonists thereofmay be therapeutically useful for anti-microbial applications. To verifythe presence of this capability in zsig32 polypeptides, agonists orantagonists of the present invention, such zsig32 polypeptides, agonistsor antagonists are evaluated with respect to their anti-microbialproperties according to procedures known in the art. See, for example,Barsum et al., Eur. Respir. J. 8: 709-14, 1995; Sandovsky-Losica et al.,J. Med. Vet. Mycol. (England) 28: 279-87, 1990; Mehentee et al., J. Gen.Microbiol (England) 135 (Pt. 8): 2181-8, 1989; Segal and Savage, J. Med.Vet. Mycol. 24: 477-9, 1986 and the like. If desired, zsig32 polypeptideperformance in this regard can be compared to proteins known to befunctional in this regard, such as proline-rich proteins, lysozyme,histatins, lactoperoxidase or the like. In addition, zsig32 polypeptidesor agonists or antagonists thereof may be evaluated in combination withone or more anti-microbial agents to identify synergistic effects.

[0074] Anti-microbial protective agents may be directly acting orindirectly acting. Such agents operating via membrane association orpore forming mechanisms of action directly attach to the offendingmicrobe. Anti-microbial agents can also act via an enzymatic mechanism,breaking down microbial protective substances or the cell wall/membranethereof. Anti-microbial agents, capable of inhibiting microorganismproliferation or action or of disrupting microorganism integrity byeither mechanism set forth herein, are useful in methods for preventingcontamination in cell culture by microbes susceptible to thatanti-microbial activity. Such techniques involve culturing cells in thepresence of an effective amount of said zsig32 polypeptide or an agonistor antagonist thereof.

[0075] Also, zsig32 polypeptides or agonists thereof may be used as cellculture reagents in in vitro studies of exogenous microorganisminfection, such as bacterial, viral or fungal infection. Such moietiesmay also be used in in vivo animal models of infection. Also, themicroorganism-adherence properties of zsig32 polypeptides or agoniststhereof can be studied under a variety of conditions in binding assaysand the like.

[0076] Moreover, zsig32 polypeptides, agonists or antagonists thereofmay be therapeutically useful for mucosal integrity maintenance. Toverify the presence of this capability in zsig32 polypeptides, agonistsor antagonists of the present invention, such zsig32 polypeptides,agonists or antagonists are evaluated with respect to their mucosalintegrity maintenance according to procedures known in the art. See, forexample, Zahm et al., Eur. Respir. J. 8: 381-6, 1995, which describesmethods for measuring viscoelastic properties and surface properties ofmucous as well as for evaluating mucous transport by cough and byciliary activity. If desired, zsig32 polypeptide performance in thisregard can be compared to mucins or the like. In addition, zsig32polypeptides or agonists or antagonists thereof may be evaluated incombination with mucins to identify synergistic effects.

[0077] In addition, a 37 amino acid peptide has been discovered which issecreted in saliva and believed to cause vasodilation of cranial bloodvessels. Such vasodilation leads to migrane attacks. This peptide isbelieved to be secreted in response to clenching of the teeth, which isalso often associated with migrane attacks. Thus, a further aspect ofthe present invention involves the determination of the vasodilatoryeffects of zsig32 polypeptides and agonists and antagonists thereof.Such determination may be made using known assay techniques. Compoundscapable of down-modulating vasodilation of blood vessels may be usefulin the prevention or treatment of migrane attacks.

[0078] Polynucleotides encoding zsig32 polypeptides are useful withingene therapy applications where it is desired to increase or inhibitzsig32 activity. If a mammal has a mutated or absent zsig32 gene, thezsig32 gene can be introduced into the cells of the mammal. In oneembodiment, a gene encoding a zsig32 polypeptide is introduced in vivoin a viral vector. Such vectors include an attenuated or defective DNAvirus, such as, but not limited to, herpes simplex virus (HSV),papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associatedvirus (AAV), and the like. Defective viruses, which entirely or almostentirely lack viral genes, are preferred. A defective virus is notinfective after introduction into a cell. Use of defective viral vectorsallows for administration to cells in a specific, localized area,without concern that the vector can infect other cells. Examples ofparticular vectors include, but are not limited to, a defective herpessimplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci.2:320-30, 1991); an attenuated adenovirus vector, such as the vectordescribed by Stratford-Perricaudet et al., J. Clin. Invest. 90:626-30,1992; and a defective adeno-associated virus vector (Samulski et al., J.Virol. 61:3096-101, 1987; Samulski et al., J. Virol. 63:3822-8, 1989).

[0079] In another embodiment, a zsig32 gene can be introduced in aretroviral vector, e.g., as described in Anderson et al., U.S. Pat. No.5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S. Pat. No.4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J.Virol. 62:1120, 1988; Temin et al., U.S. Pat. No. 5,124,263;International Patent Publication NO: WO 95/07358, published Mar. 16,1995 by Dougherty et al.; and Kuo et al., Blood 82:845, 1993.Alternatively, the vector can be introduced by lipofection in vivo usingliposomes. Synthetic cationic lipids can be used to prepare liposomesfor in vivo transfection of a gene encoding a marker (Felgner et al.,Proc. Natl. Acad. Sci. USA 84:7413-7, 1987; Mackey et al., Proc. Natl.Acad. Sci. USA 85:8027-31, 1988). The use of lipofection to introduceexogenous genes into specific organs in vivo has certain practicaladvantages. Molecular targeting of liposomes to specific cellsrepresents one area of benefit. More particularly, directingtransfection to particular cells represents one area of benefit. Forinstance, directing transfection to particular cell types would beparticularly advantageous in a tissue with cellular heterogeneity, suchas the pancreas, liver, kidney, and brain. Lipids may be chemicallycoupled to other molecules for the purpose of targeting. Targetedpeptides (e.g., hormones or neurotransmitters), proteins such asantibodies, or non-peptide molecules can be coupled to liposomeschemically.

[0080] It is possible to remove the target cells from the body; tointroduce the vector as a naked DNA plasmid; and then to re-implant thetransformed cells into the body. Naked DNA vectors for gene therapy canbe introduced into the desired host cells by methods known in the art,e.g., transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a gene gunor use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem.267:963-7, 1992; Wu et al., J. Biol. Chem. 263:14621-4,. 1988.

[0081] Antisense methodology can be used to inhibit zsig32 genetranscription, such as to inhibit cell proliferation in vivo.Polynucleotides that are complementary to a segment of a zsig32-encodingpolynucleotide (e.g., a polynucleotide as set froth in SEQ ID NO:1) aredesigned to bind to zsig32-encoding mRNA and to inhibit translation ofsuch mRNA. Such antisense polynucleotides are used to inhibit expressionof zsig32 polypeptide-encoding genes in cell culture or in a subject.

[0082] Transgenic mice, engineered to express the zsig32 gene, and micethat exhibit a complete absence of zsig32 gene function, referred to as“knockout mice” (Snouwaert et al., Science 257:1083, 1992), may also begenerated (Lowell et al., Nature 366:740-42, 1993). These mice may beemployed to study the zsig32 gene and the protein encoded thereby in anin vivo system.

[0083] The present invention also provides reagents for use indiagnostic applications. For example, the zsig32 gene, a probecomprising zsig32 DNA or RNA, or a subsequence thereof can be used todetermine if the zsig32 gene is present on chromosome 16 or if amutation has occurred. Detectable chromosomal aberrations at the zsig32gene locus include, but are not limited to, aneuploidy, gene copy numberchanges, insertions, deletions, restriction site changes andrearrangements. These aberrations can occur within the coding sequence,within introns, or within flanking sequences, including upstreampromoter and regulatory regions, and may be manifested as physicalalterations within a coding sequence or changes in gene expressionlevel.

[0084] In general, these diagnostic methods comprise the steps of (a)obtaining a genetic sample from a patient; (b) incubating the geneticsample with a polynucleotide probe or primer as disclosed above, underconditions wherein the polynucleotide will hybridize to complementarypolynucleotide sequence, to produce a first reaction product; and (iii)comparing the first reaction product to a control reaction product. Adifference between the first reaction product and the control reactionproduct is indicative of a genetic abnormality in the patient. Geneticsamples for use within the present invention include genomic DNA, cDNA,and RNA. The polynucleotide probe or primer can be RNA or DNA, and willcomprise a portion of SEQ ID NO:1, the complement of SEQ ID NO:1, or anRNA equivalent thereof. Suitable assay methods in this regard includemolecular genetic techniques known to those in the art, such asrestriction fragment length polymorphism (RFLP) analysis, short tandemrepeat (STR) analysis employing PCR techniques, ligation -chain reaction(Barany, PCR Methods and Applications 1:5-16, 1991), ribonucleaseprotection assays, and other genetic linkage analysis techniques knownin the art (Sambrook et al., ibid.; Ausubel et. al., ibid.; Marian,Chest 108:255-65, 1995). Ribonuclease protection assays (see, e.g.,Ausubel et al., ibid., ch. 4) comprise the hybridization of an RNA probeto a patient RNA sample, after which the reaction product (RNA-RNAhybrid) is exposed to RNase. Hybridized regions of the RNA are protectedfrom digestion. Within PCR assays, a patient's genetic sample isincubated with a pair of polynucleotide primers, and the region betweenthe primers is amplified and recovered. Changes in size or amount ofrecovered product are indicative of mutations in the patient. AnotherPCR-based technique that can be employed is single strand conformationalpolymorphism (SSCP) analysis (Hayashi, PCR Methods and Applications1:34-8, 1991).

[0085] Within another aspect of the present invention there is provideda pharmaceutical composition comprising purified zsig32 polypeptide incombination with a pharmaceutically acceptable vehicle. Suchpharmaceutical compositions may be administered to prevent or treatsalivary gland dysfunction. Such prevention or treatment may be directedto digestive dysfunction, such as a deficiency in starch breakdowncapability or efficiency, wound healing dysfunction, inadequate salivaproduction or composition or mucosal integrity breakdown. Zsig32polypeptides may also have an anti-microbial function, most likelystemming from an exposed adhesion motif as discussed herein. Also,expression of zsig32 polypeptide at a relatively high level in tracheamay indicate a role for zsig32 polypeptides in prevention or treatmentof destructive lung disease. Examples of pathological conditions,characterized by one or more of the aforementioned criteria, includexerostomia, sarcoidosis, dental caries, osteomyelitis, oral candidiasis,buccal mucosa infections, chronic inflammation (Sjogren's syndrome),mumps, chronic bronchitis, adult respiratory distress syndrome (ARDS),sudden infant death syndrome (SIDS), salivary gland carcinoma,pneumocystic carinii (particularly as associated with AIDS patients),cystic fibrosis, emphysema and the like.

[0086] Evaluation of zsig32 polypeptide involvement in such conditionsmay be conducted using in vivo or in vitro methods that are known tothose of ordinary skill in the art. For example, bronchoalveolar lavagemay be employed in the assessment of destructive lung diseases, such aspulmonary emphysema, chronic bronchitis, cystic fibrosis, ARDS and thelike. See, for example, Luisetti et al., Respiration 59(suppl. 1): 24-7,1992. Salivary gland, lacrimal gland and labial salivary gland biopsesmay be employed in the evaluation of xerostomia. See, for example,Matsumoto et al., J. Clin. Invest. 97(8): 1969-77, 1996. This calciumchannel dependent condition has also been evaluated using fura-2 assaysof intracellular calcium ion concentration, as described in Seagrave etal., Archs. Oral Biol. 41(5): 425-30, 1996. Alymphoplasia (aly) mice area useful animal model for systemic Sjogren's syndrome, an autoimmunedisease characterized by lymphocytic infiltration into the lachrymal andsalivary glands, leading to symptomatic dry eyes and mouth. See, forexample, Furukawa et al., Brit. J. Rheum. 35: 1223-30, 1996.

[0087] The present invention provides methods for identifying agonistsor antagonists of the zsig32 polypeptides disclosed herein, whichagonists or antagonists may have valuable therapeutic properties asdiscussed further herein. Within one embodiment, there is provided amethod of identifying zsig32 polypeptide agonists, comprising providingcells responsive to a zsig32 polypeptide as disclosed herein, culturingthe cells in the presence of a test compound and comparing the cellularresponse with the cell cultured in the presence of the zsig32polypeptide, and selecting the test compounds for which the cellularresponse is of the same type. Agonists are therefore useful to mimic oraugment the function of zsig32 polypeptides.

[0088] Within another embodiment, there is provided a method ofidentifying antagonists of zsig32 polypeptide, comprising providingcells responsive to a zsig32 polypeptide, culturing a first portion ofthe cells in the presence of zsig32 polypeptide, culturing a secondportion of the cells in the presence of the zsig32 polypeptide and atest compound, and detecting a decrease in a cellular response of thesecond portion of the cells as compared to the first portion of thecells. Antagonists are therefore useful to inhibit or diminish zsig32polypeptide function.

[0089] Zsig32 polypeptides of the present invention exhibit an adhesionmotif at amino acid residues 63-65 of SEQ ID NO: 2. According tothree-dimensional analysis of polypeptide structure, the adhesion motifappears to be exposed. Thus, zsig32 polypeptides, fragments thereofincorporating such an adhesion motif or fusion proteins incorporatingzsig32 polypeptide or the adhesion motif-containing fragment thereofappear to be useful in the study of adhesion. Such study can beaccomplished using a method of modulating adhesion of platelets, forexample, in cell culture, comprising incubating platelets in a culturemedium comprising a zsig32 polypeptide as disclosed above in an amountsufficient to modulate adhesion. Assays for evaluation of plateletadhesion are known in the art.

[0090] Zsig32 polypeptides or antagonists or agonists thereof areexpected be useful in circumstances where modulation of adhesion isdesired. Such adhesion-modulating function may be used in in vitroexperiments designed to study adhesion, such as adhesion ofmicroorganisms to cells, tissue or mucous. Enhancers and inhibitors ofadhesion also have potential as therapeutics for conditions requiringsuch modulation. For example, enhanced tumor cell-tumor cell adhesion ina primary solid tumor does not favor metastasis thereof. Also,diminished tumor cell-endothelial cell adhesion also does not favormetastasis formation at a site distant from the primary tumor. Assays toassess metastatic potential, assessed using adhesion parameters, areknown in the art. See, for example, Koenigsmann et al., Onkologie 17:528-37, 1994, Asao et al., Canc. Letts. 78: 57-62, 1994 and the like.

[0091] Preliminary analysis of mice receiving intravenous tail veininjections of adenovirus expressing a zsig32 polypeptides suggests thatzsig32 may increase platelet levels 25-30% above normal. Cytokines suchas erythropoietin and IL-6 also increase platelet levels by 25-30%.Therapeutic application of such activity would be useful wherever it isdesirable to increase proliferation of such cells, such as bone marrowtransplantation, in the treatment of cytopenia, such as that induced byaplastic anemia, myelodisplastic syndromes, chemotherapy or congenitalcytopenias. Application could also be made in the treatment ofthrombocytopenia. Use of proteins capable of platelet stimulation wouldalso be valuable as tools for the in vitro study of the differentiationand development of hematopoietic cells, such as for elucidating themechanisms of cell differentiation and for determining the lineages ofmature cells, and may also find utility as proliferative agents in cellculture. Platelets are directed to sites of injuries and are believed tobe mediators of wound healing and, under some circumstances, mediatorsof pathogenesis.

[0092] A still further aspect of the invention provides useful researchreagents and methods for evaluating salivary gland function. Zsig32polypeptides, agonists or antagonists thereof can be admixed with testsaliva or one or more proteins contained in saliva to provide cultureconditions under which salivary gland function can be studied. Forexample, admixture of zsig32 polypeptides, agonists or antagoniststhereof can be combined with one or more growth factors to provide aculture medium in which the wound healing properties of saliva can bestudied.

[0093] Within preferred embodiments of the invention the isolatedpolynucleotides will hybridize to similar sized regions of SEQ ID NO: 2,SEQ ID NO: 6, SEQ ID NO: 10, or SEQ ID NO: 11, the other specific probesreferred to herein, or a sequence complementary thereto, under stringentconditions. In general, stringent conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Typicalstringent conditions are those in which the salt concentration is lessthan about 0.02 M at pH 7 and the temperature is at least about 60° C.

[0094] As previously noted, the isolated zsig32 polynucleotides of thepresent invention include DNA and RNA. Methods for isolating DNA and RNAare well known in the art. It is generally preferred to isolate RNA fromsalivary gland tissues, although DNA can also be prepared using RNA fromother tissues or isolated as genomic DNA. Total RNA can be preparedusing guanidine HCl extraction followed by isolation by centrifugationin a CsCl gradient (Chirgwin et al., Biochemistry 18:52-94, 1979). Poly(A)⁺ RNA is prepared from total RNA using the method of Aviv and Leder(Proc. Natl. Acad. Sci. USA 69:1408-12, 1972). Complementary DNA (cDNA)is prepared from poly(A)⁺ RNA using known methods. Polynucleotidesencoding zsig32 polypeptides are then identified and isolated by, forexample, hybridization or PCR.

[0095] The present invention further provides counterpart polypeptidesand polynucleotides from other species (orthologs). These speciesinclude, but are not limited to mammalian, avian, amphibian, reptile,fish, insect and other vertebrate and invertebrate species. Ofparticular interest are zsig32 polypeptides from other mammalianspecies, including murine, rat, porcine, ovine, bovine, canine, feline,equine and other primate proteins. Species homologs of the humanproteins can be cloned using information and compositions provided bythe present invention in combination with conventional cloningtechniques. For example, a cDNA can be cloned using mRNA obtained from atissue or cell type that expresses the protein. Suitable sources of mRNAcan be identified by probing Northern blots with probes designed fromthe sequences disclosed herein. A library is then prepared from mRNA ofa positive tissue of cell line. A zsig32-encoding cDNA can then beisolated by a variety of methods, such as by probing with a complete orpartial human cDNA or with one or more sets of degenerate probes basedon the disclosed sequences. A cDNA can also be cloned using thepolymerase chain reaction, or PCR (Mullis, U.S. Pat. No. 4,683,202),using primers designed from the sequences disclosed herein. Within anadditional method, the cDNA library can be used to transform ortransfect host cells, and expression of the cDNA of interest can bedetected with an antibody to an epitope of a zsig32 polypeptide. Similartechniques can also be applied to the isolation of genomic clones.

[0096] Those skilled in the art will recognize that the sequencesdisclosed in SEQ ID NO:1 and SEQ ID NO:2 represent a single allele ofthe human zsig32 gene and polypeptide, and that allelic variation andalternative splicing are expected to occur. Allelic variants can becloned by probing cDNA or genomic libraries from different individualsaccording to standard procedures. Allelic variants of the DNA sequenceshown in SEQ ID NO: 1, including those containing silent mutations andthose in which mutations result in amino acid sequence changes, arewithin the scope of the present invention, as are proteins which areallelic variants of SEQ ID NO:2. cDNAs generated from alternativelyspliced mRNAs, which retain the properties of the zsig32 polypeptide areincluded within the scope of the present invention, as are polypeptidesencoded by such cDNAs and mRNAs. Allelic variants and splice variants ofthese sequences can be cloned by probing cDNA or genomic libraries fromdifferent individuals or tissues according to standard procedures knownin the art.

[0097] The present invention also provides isolated zsig32 polypeptidesthat are substantially homologous to the polypeptides of SEQ ID NO:2 andtheir orthologs. The term “substantially homologous” is used herein todenote polypeptides having 50%, preferably 60%, more preferably at least80%, sequence identity to the sequences shown in SEQ ID NO:2 or theirorthologs. Such polypeptides will more preferably be at least 90%identical, and most preferably 95% or more identical to SEQ ID NO:2 orits orthologs. Percent sequence identity is determined by conventionalmethods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16,1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-9,1992. Briefly, two amino acid sequences are aligned to optimize thealignment scores using a gap opening penalty of 10, a gap extensionpenalty of 1, and the “blosum 62” scoring matrix of Henikoff andHenikoff (ibid.) as shown in Table 3 (amino acids are indicated by thestandard one-letter codes). The percent identity is then calculated as:$\frac{\text{Total~~number~~of~~identical~~matches}}{\begin{matrix}\begin{matrix}\begin{matrix}\text{[length~~of~~the~~longer~~sequence~~plus~~the} \\\text{number~~of~~gaps~~introduced~~into~~the}\end{matrix} \\\text{longer~~sequence~~in~~order~~to~~align}\end{matrix} \\\text{the~~two~~sequences]}\end{matrix}} \times 100$

TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

[0098] Sequence identity of polynucleotide molecules is determined bysimilar methods using a ratio as disclosed above.

[0099] Substantially homologous proteins and polypeptides arecharacterized as having one or more amino acid substitutions, deletionsor additions. These changes are preferably of a minor nature, that isconservative amino acid substitutions (see Table 4) and othersubstitutions that do not significantly affect the folding or activityof the protein or polypeptide; small deletions, typically of one toabout 30 amino acids; and small amino- or carboxyl-terminal extensions,such as an amino-terminal methionine residue, a small linker peptide ofup to about 20-25 residues, or an affinity tag. Polypeptides comprisingaffinity tags can further comprise a proteolytic cleavage site betweenthe zsig32 polypeptide and the affinity tag. Preferred such sitesinclude thrombin cleavage sites and factor Xa cleavage sites. TABLE 4Conservative amino acid substitutions Basic: arginine lysine histidineAcidic: glutamic acid aspartic acid Polar: glutamine asparagineHydrophobic: leucine isoleucine valine Aromatic: phenylalaninetryptophan tyrosine Small: glycine alanine serine threonine methionine

[0100] The present invention further provides a variety of otherpolypeptide fusions [and related multimeric proteins comprising one ormore polypeptide fusions]. For example, a zsig32 polypeptide can beprepared as a fusion to a dimerizing protein as disclosed in U.S. Pat.Nos. 5,155,027 and 5,567,584. Preferred dimerizing proteins in thisregard include immunoglobulin constant region domains.Immunoglobulin-zsig32 polypeptide fusions can be expressed ingenetically engineered cells [to produce a variety of multimeric zsig32analogs]. Auxiliary domains can be fused to zsig32 polypeptides totarget them to specific cells, tissues, or macromolecules. For example,a zsig32 polypeptide or protein could be targeted to a predeterminedcell type by fusing a zsig32 polypeptide to a ligand that specificallybinds to a receptor on the surface of the target cell. In this way,polypeptides and proteins can be targeted for therapeutic or diagnosticpurposes. A zsig32 polypeptide can be fused to two or more moieties,such as an affinity tag for purification and a targeting domain.Polypeptide fusions can also comprise one or more cleavage sites,particularly between domains. See, Tuan et al., Connect. Tiss. Res.34:1-9, 1996.

[0101] The proteins of the present invention can also comprisenon-naturally occurring amino acid residues. Non-naturally occurringamino acids include, without limitation, trans-3-methylproline,2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline,N-methyl-glycine, allo-threonine, methylthreonine,hydroxyethyl-cysteine, hydroxyethylhomocysteine, nitroglutamine,homo-glutamine, pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, 3,3-dimethyl-proline,tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, and 4-fluorophenyl-alanine. Several methods areknown in the art for incorporating non-naturally occurring amino acidresidues into proteins. For example, an in vitro system can be employedwherein nonsense mutations are suppressed using chemically aminoacylatedsuppressor tRNAs. Methods for synthesizing amino acids andaminoacylating tRNA are known in the art. Transcription and translationof plasmids containing nonsense mutations is carried out in a cell-freesystem comprising an E. coli S30 extract and commercially availableenzymes and other reagents. Proteins are purified by chromatography.See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991;Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA90:10145-9, 1993). In a second method, translation is carried out inXenopus oocytes by microinjection of mutated mRNA and chemicallyaminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.271:19991-8, 1996). Within a third method, E. coli cells are cultured inthe absence of a natural amino acid that is to be replaced (e.g.,phenylalanine) and in the presence of the desired non-naturallyoccurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturallyoccurring amino acid is incorporated into the protein in place of itsnatural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994.Naturally occurring amino acid residues can be converted tonon-naturally occurring species by in vitro chemical modification.Chemical modification can be combined with site-directed mutagenesis tofurther expand the range of substitutions (Wynn and Richards, ProteinSci. 2:395-403, 1993).

[0102] A limited number of non-conservative amino acids, amino acidsthat are not encoded by the genetic code, non-naturally occurring aminoacids, and unnatural amino acids may be substituted for zsig32 aminoacid residues.

[0103] Essential amino acids in the zsig32 polypeptides of the presentinvention can be identified according to procedures known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244: 1081-5, 1989). In the lattertechnique, single alanine mutations are introduced at every residue inthe molecule, and the resultant mutant molecules are tested forbiological activity (e.g., androgen regulation, anti-microbial activity,adhesion modulation or the like) to identify amino acid residues thatare critical to the activity of the molecule. See also, Hilton et al.,J. Biol. Chem. 271:4699-708, 1996. Sites of ligand-receptor interactioncan also be determined by physical analysis of structure, as determinedby such techniques as nuclear magnetic resonance, crystallography,electron diffraction or photoaffinity labeling, in conjunction withmutation of putative contact site amino acids. See, for example, de Voset al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol.224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. Theidentities of essential amino acids can also be inferred from analysisof homologies with related proteins.

[0104] Multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner etal., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Neret al., DNA 7:127, 1988).

[0105] Variants of the disclosed zsig32 DNA and polypeptide sequencescan be generated through DNA shuffling as disclosed by Stemmer, Nature370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-51, 1994and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated byin vitro homologous recombination by random fragmentation of a parentDNA followed by reassembly using PCR, resulting in randomly introducedpoint mutations. This technique can be modified by using a family ofparent DNAs, such as allelic variants or DNAs from different species, tointroduce additional variability into the process. Selection orscreening for the desired activity, followed by additional iterations ofmutagenesis and assay provides for rapid “evolution” of sequences byselecting for desirable mutations while simultaneously selecting againstdetrimental changes.

[0106] Mutagenesis methods as disclosed above can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode active polypeptides (e.g., those susceptible toandrogen regulation, capable of anti-microbial action or adhesionmodulation or the like) can be recovered from the host cells and rapidlysequenced using modern equipment. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide of interest, and can be applied to polypeptides of unknownstructure.

[0107] Using the methods discussed herein, one of ordinary skill in theart can identify and/or prepare a variety of polypeptides that aresubstantially homologous to residues 23 (Gly) to 178 (Arg) of SEQ ID NO:2 or allelic variants thereof and retain the one or more properties ofthe wild-type protein. Such polypeptides may include additional aminoacids, such as affinity tags or the like. Such polypeptides may alsoinclude additional polypeptide segments as generally discussed herein.

[0108] The polypeptides of the present invention, including full-lengthproteins, fragments thereof and fusion proteins, can be produced ingenetically engineered host cells according to conventional techniques.Suitable host cells are those cell types that can be transformed ortransfected with exogenous DNA and grown in culture, and includebacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryoticcells, particularly cultured cells of multicellular organisms, arepreferred. Techniques for manipulating cloned DNA molecules andintroducing exogenous DNA into a variety of host cells are disclosed bySambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, andAusubel et al. (eds.), Current Protocols in Molecular Biology, JohnWiley and Sons, Inc., NY, 1987.

[0109] In general, a DNA sequence encoding a zsig32 polypeptide of thepresent invention is operably linked to other genetic elements requiredfor its expression, generally including a transcription promoter andterminator within an expression vector. The vector will also commonlycontain one or more selectable markers and one or more origins ofreplication, although those skilled in the art will recognize thatwithin certain systems selectable markers may be provided on separatevectors, and replication of the exogenous DNA may be provided byintegration into the host cell genome. Selection of promoters,terminators, selectable markers, vectors and other elements is a matterof routine design within the level of ordinary skill in the art. Manysuch elements are described in the literature and are available throughcommercial suppliers.

[0110] To direct a zsig32 polypeptide into the secretory pathway of ahost cell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of the zsig32 polypeptide, ormay be derived from another secreted protein (e.g., t-PA) or synthesizedde novo. The secretory signal sequence is joined to the zsig32-encodingDNA sequence in the correct reading frame. Secretory signal sequencesare commonly positioned 5′ to the DNA sequence encoding the polypeptideof interest, although certain signal sequences may be positionedelsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S.Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).Conversely, the signal sequence portion of the zsig32 polypeptide (aminoacids 1-22 or 7-22 of SEQ ID NO: 2) may be employed to direct thesecretion of an alternative protein by analogous methods.

[0111] Cultured mammalian cells are also preferred hosts within thepresent invention. Methods for introducing exogenous DNA into mammalianhost cells include calcium phosphate-mediated transfection (Wigler etal., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics7:603, 1981: Graham and Van der Eb, Virology 52:456, 1973),electroporation (Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextranmediated transfection (Ausubel et al., eds., Current Protocols inMolecular Biology, John Wiley and Sons, Inc., NY, 1987),liposome-mediated transfection (Hawley-Nelson et al., Focus 15:73, 1993;Ciccarone et al., Focus 15:80, 1993), and viral vectors (Miller andRosman, BioTechniques 7:980-90, 1989; Wang and Finer, Nat. Med.2:714-16, 1996). The production of recombinant polypeptides in culturedmammalian cells is disclosed, for example, by Levinson et al., U.S. Pat.No. 4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al.,U.S. Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134. Suitablecultured mammalian cells include the COS-1 (ATCC NO: CRL 1650), COS-7(ATCC NO: CRL 1651), BHK 570 (ATCC NO: CRL 10314), 293 (ATCC NO: CRL1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamsterovary (e.g. CHO-K1; ATCC NO: CCL 61) cell lines. Additional suitablecell lines are known in the art and available from public depositoriessuch as the American Type Culture Collection, Rockville, Md. In general,strong transcription promoters are preferred, such as promoters fromSV-40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Othersuitable promoters include those from metallothionein genes (U.S. Pat.Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

[0112] Drug selection is generally used to select for cultured mammaliancells into which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems mayalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g., hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that introducean altered phenotype, such as green fluorescent protein, or cell surfaceproteins such as CD4, CD8, Class I MHC, placental alkaline phosphatasemay be used to sort transfected cells from untransfected cells by suchmeans as FACS sorting or magnetic bead separation technology.

[0113] Other higher eukaryotic cells can also be used as hosts,including insect cells, plant cells and avian cells. The use ofAgrobacterium rhizogenes as a vector for expressing genes in plant cellshas been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58,1987. Transformation of insect cells and production of foreignpolypeptides therein is disclosed by Guarino et al., U.S. Pat. No.5,162,222; Bang et al., U.S. Pat. No. 4,775,624; and WIPO publication WO94/06463. Insect cells can be infected with recombinant baculovirus,commonly derived from Autographa californica nuclear polyhedrosis virus(AcNPV). DNA encoding the zsisg32 polypeptide is inserted into thebaculoviral genome in place of the AcNPV polyhedrin gene coding sequenceby one of two methods. The first is the traditional method of homologousDNA recombination between wild-type AcNPV and a transfer vectorcontaining the zsig32 flanked by AcNPV sequences. Suitable insect cells,e.g. SF9 cells, are infected with wild-type AcNPV and transfected with atransfer vector comprising a zsig32 polynucleotide operably linked to anAcNPV polyhedrin gene promoter, terminator, and flanking sequences. See,King, and Possee, The Baculovirus Expression System: A Laboratory Guide,London, Chapman & Hall; O'Reilly et al., Baculovirus Expression Vectors:A Laboratory Manual, New York, Oxford University Press., 1994; and,Richardson, Ed., Baculovirus Expression Protocols. Methods in MolecularBiology, Totowa, N.J., Humana Press, 1995. Natural recombination withinan insect cell will result in a recombinant baculovirus which containszsig32 driven by the polyhedrin promoter. Recombinant viral stocks aremade by methods commonly used in the art.

[0114] The second method of making recombinant baculovirus utilizes atransposon-based system described by Luckow (Luckow, et al., J. Virol.67:4566-79, 1993). This system is sold in the Bac-to-Bac kit (LifeTechnologies, Rockville, Md.). This system utilizes a transfer vector,pFastBacl™ (Life Technologies) containing a Tn7 transposon to move theDNA encoding the zsig32 polypeptide into a baculovirus genome maintainedin E. coli as a large plasmid called a “bacmid.” The pFastBacl™ transfervector utilizes the AcNPV polyhedrin promoter to drive the expression ofthe gene of interest, in this case zsig32. However, pFastBac1™ can bemodified to a considerable degree. The polyhedrin promoter can beremoved and substituted with the baculovirus basic protein promoter(also known as Pcor, p6.9 or MP promoter) which is expressed earlier inthe baculovirus infection, and has been shown to be advantageous forexpressing secreted proteins. See, Hill-Perkins and Possee, J. Gen.Virol. 71:971-6, 1990; Bonning et al., J. Gen. Virol. 75:1551-6, 1994;and, Chazenbalk and Rapoport, J. Biol. Chem. 270:1543-9, 1995. In suchtransfer vector constructs, a short or long version of the basic proteinpromoter can be used. Moreover, transfer vectors can be constructedwhich replace the native zsig32 secretory signal sequences withsecretory signal sequences derived from insect proteins. For example, asecretory signal sequence from Ecdysteroid Glucosyltransferase (EGT),honey bee Melittin (Invitrogen, Carlsbad, Calif.), or baculovirus gp67(PharMingen, San Diego, Calif.) can be used in constructs to replace thenative zsig32 secretory signal sequence. In addition, transfer vectorscan include an in-frame fusion with DNA encoding an epitope tag at theC- or N-terminus of the expressed zsig32 polypeptide, for example, aGlu-Glu epitope tag (Grussenmeyer et al., ibid.). Using techniques knownin the art, a transfer vector containing zsig32 is transformed into E.coli, and screened for bacmids which contain an interrupted lacZ geneindicative of recombinant baculovirus. The bacmid DNA containing therecombinant baculovirus genome is isolated, using common techniques, andused to transfect Spodoptera frugiperda cells, e.g. Sf9 cells.Recombinant virus that expresses zsig32 is subsequently produced.Recombinant viral stocks are made by methods commonly used the art.

[0115] The recombinant virus is used to infect host cells, typically acell line derived from the fall armyworm, Spodoptera frugiperda. See, ingeneral, Glick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA, ASM Press, Washington, D.C., 1994.Another suitable cell line is the High FiveO™ cell line (Invitrogen)derived from Trichoplusia ni (U.S. Pat. No. 5,300,435). Commerciallyavailable serum-free media are used to grow and maintain the cells.Suitable media are Sf900 II™ (Life Technologies) or ESF 921™ (ExpressionSystems) for the Sf9 cells; and Ex-cellO405™ (JRH Biosciences, Lenexa,Kans.) or Express FiveO™ (Life Technologies) for the T. ni cells. Thecells are grown up from an inoculation density of approximately 2-5×10⁵cells to a density of 1-2×10⁶ cells at which time a recombinant viralstock is added at a multiplicity of infection (MOI) of 0.1 to 10, moretypically near 3. The recombinant virus-infected cells typically producethe recombinant zsig32 polypeptide at 12-72 hours post-infection andsecrete it with varying efficiency into the medium. The culture isusually harvested 48 hours post-infection. Centrifugation is used toseparate the cells from the medium (supernatant). The supernatantcontaining the zsig32 polypeptide is filtered through micropore filters,usually 0.45 μm pore size. Procedures used are generally described inavailable laboratory manuals (King and Possee, ibid.; O'Reilly, et al.,ibid.; Richardson, ibid.). Subsequent purification of the zsig32polypeptide from the supernatant can be achieved using methods describedherein.

[0116] Fungal cells, including yeast cells, and particularly cells ofthe genus Saccharomyces, can also be used within the present invention,such as for producing zsig32 polypeptide fragments or polypeptidefusions. Methods for transforming yeast cells with exogenous DNA andproducing recombinant polypeptides therefrom are disclosed by, forexample, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat.No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat.No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformedcells are selected by phenotype determined by the selectable marker,commonly drug resistance or the ability to grow in the absence of aparticular nutrient (e.g., leucine). A preferred vector system for usein yeast is the POT1 vector system disclosed by Kawasaki et al. (U.S.Pat. No. 4,931,373), which allows transformed cells to be selected bygrowth in glucose-containing media. Suitable promoters and terminatorsfor use in yeast include those from glycolytic enzyme genes (see, e.g.,Kawasaki, U.S. Pat. No. 4,599,311; Kingsman et al., U.S. Pat. No.4,615,974; and Bitter, U.S. Pat. No. 4,977,092) and alcoholdehydrogenase genes. See also U.S. Pat. Nos. 4,990,446; 5,063,154;5,139,936 and 4,661,454. Transformation systems for other yeasts,including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyceslactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichiamethanolica, Pichia guillermondlii and Candida maltosa are known in theart. See, for example, Gleeson et al., J. Gen. Microbiol. 132:3459-3465,1986 and Cregg, U.S. Pat. No. 4,882,279. Aspergillus cells may beutilized according to the methods of McKnight et al., U.S. Pat. No.4,935,349. Methods for transforming Acremonium chrysogenum are disclosedby Sumino et al., U.S. Pat. No. 5,162,228. Methods for transformingNeurospora are disclosed by Lambowitz, U.S. Pat. No. 4,486,533.

[0117] The use of Pichia methanolica as host for the production ofrecombinant proteins is disclosed in WIPO Publications WO 97/17450, WO97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use intransforming P. methanolica will commonly be prepared asdouble-stranded, circular plasmids, which are preferably linearizedprior to transformation. For polypeptide production in P. methanolica,it is preferred that the promoter and terminator in the plasmid be thatof a P. methanolica gene, such as a P. methanolica alcohol utilizationgene (AUG1 or AUG2). Other useful promoters include those of thedihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), andcatalase (CAT) genes. To facilitate integration of the DNA into the hostchromosome, it is preferred to have the entire expression segment of theplasmid flanked at both ends by host DNA sequences. A preferredselectable marker for use in Pichia methanolica is a P. methanolica ADE2gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC;EC 4.1.1.21), which allows ade2 host cells to grow in the absence ofadenine. For large-scale, industrial processes where it is desirable tominimize the use of methanol, it is preferred to use host cells in whichboth methanol utilization genes (AUG1 and AUG2) are deleted. Forproduction of secreted proteins, host cells deficient in vacuolarprotease genes (PEP4 and PRB1) are preferred. Electroporation is used tofacilitate the introduction of a plasmid containing DNA encoding apolypeptide of interest into P. methanolica cells. It is preferred totransform P. methanolica cells by electroporation using an exponentiallydecaying, pulsed electric field having a field strength of from 2.5 to4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (τ) of from1 to 40 milliseconds, most preferably about 20 milliseconds.

[0118] Prokaryotic host cells, including strains of the bacteriaEscherichia coli, Bacillus and other genera are also useful host cellswithin the present invention. Techniques for transforming these hostsand expressing foreign DNA sequences cloned therein are well known inthe art (see, e.g., Sambrook et al., ibid.). When expressing a zsig32polypeptide in bacteria such as E. coli, the polypeptide may be retainedin the cytoplasm, typically as insoluble granules, or may be directed tothe periplasmic space by a bacterial secretion sequence. In the formercase, the cells are lysed, and the granules are recovered and denaturedusing, for example, guanidine isothiocyanate or urea. The denaturedpolypeptide can then be refolded and dimerized by diluting thedenaturant, such as by dialysis against a solution of urea and acombination of reduced and oxidized glutathione, followed by dialysisagainst a buffered saline solution. In the latter case, the polypeptidecan be recovered from the periplasmic space in a soluble and functionalform by disrupting the cells (by, for example, sonication or osmoticshock) to release the contents of the periplasmic space and recoveringthe protein, thereby obviating the need for denaturation and refolding.

[0119] Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. P. methanolicacells are cultured in a medium comprising adequate sources of carbon,nitrogen and trace nutrients at a temperature of about 25° C. to 35° C.Liquid cultures are provided with sufficient aeration by conventionalmeans, such as shaking of small flasks or sparging of fermentors. Apreferred culture medium for P. methanolica is YEPD (2% D-glucose, 2%Bacto™ Peptone (Difco Laboratories, Detroit, Mich.), 1% Bacto™ yeastextract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).

[0120] Expressed recombinant zsig32 polypeptides (or chimeric zsig32polypeptides) can be purified using fractionation and/or conventionalpurification methods and media. Ammonium sulfate precipitation and acidor chaotrope extraction may be used for fractionation of samples.Exemplary purification steps may include hydroxyapatite, size exclusion,FPLC and reverse-phase high performance liquid chromatography. Suitablechromatographic media include derivatized dextrans, agarose, cellulose,polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Qderivatives are preferred, with DEAE Fast-Flow Sepharose (Pharmacia,Piscataway, N.J.) being particularly preferred. Exemplarychromatographic media include those media derivatized with phenyl,butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia),Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose(Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG71 (Toso Haas) and the like. Suitable solid supports include glassbeads, silica-based resins, cellulosic resins, agarose beads,cross-linked agarose beads, polystyrene beads, cross-linkedpolyacrylamide resins and the like that are insoluble under theconditions in which they are to be used. These supports may be modifiedwith reactive groups that allow attachment of proteins by amino groups,carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydratemoieties. Examples of coupling chemistries include cyanogen bromideactivation, N-hydroxysuccinimide activation, epoxide activation,sulfhydryl activation, hydrazide activation, and carboxyl and aminoderivatives for carbodiimide coupling chemistries. These and other solidmedia are well known and widely used in the art, and are available fromcommercial suppliers. Methods for binding receptor polypeptides tosupport media are well known in the art. Selection of a particularmethod is a matter of routine design and is determined in part by theproperties of the chosen support. See, for example, AffinityChromatography: Principles & Methods, Pharmacia LKB Biotechnology,Uppsala, Sweden, 1988.

[0121] The polypeptides of the present invention can be isolated byexploitation of their structural properties. For example, immobilizedmetal ion adsorption (IMAC) chromatography can be used to purifyhistidine-rich proteins, including those comprising polyhistidine tags.Briefly, a gel is first charged with divalent metal ions to form achelate (Sulkowski, Trends in Biochem. 3:1-7, 1985). Histidine-richproteins will be adsorbed to this matrix with differing affinities,depending upon the metal ion used, and will be eluted by competitiveelution, lowering the pH, or use of strong chelating agents. Othermethods of purification include purification of glycosylated proteins bylectin affinity chromatography and ion exchange chromatography (Methodsin Enzymol., Vol. 182, “Guide to Protein Purification”, M. Deutscher,(ed.), Acad. Press, San Diego, 1990, pp.529-39). Within additionalembodiments of the invention, a fusion of the polypeptide of interestand an affinity tag (e.g., maltose-binding protein, Glu-Glu, animmunoglobulin domain) may be constructed to facilitate purification.

[0122] Protein refolding (and optionally reoxidation) procedures may beadvantageously used. It is preferred to purify the protein to >80%purity, more preferably to >90% purity, even more preferably >95%, andparticularly preferred is a pharmaceutically pure state, that is greaterthan 99.9% pure with respect to contaminating macromolecules,particularly other proteins and nucleic acids, and free of infectiousand pyrogenic agents. Preferably, a purified protein is substantiallyfree of other proteins, particularly other proteins of animal origin.

[0123] Zsig32 polypeptides or fragments thereof may also be preparedthrough chemical synthesis. Zsig32 polypeptides may be monomers ormultimers; glycosylated or non-glycosylated; pegylated or non-pegylated;and may or may not include an initial methionine amino acid residue.

[0124] A zsig32 polypeptide binding protein can also be used inpurification applications. The binding protein is immobilized on a solidsupport, such as beads of agarose, cross-linked agarose, glass,cellulosic resins, silica-based resins, polystyrene, cross-linkedpolyacrylamide, or like materials that are stable under the conditionsof use. Methods for linking polypeptides to solid supports are known inthe art, and include amine chemistry, cyanogen bromide activation,N-hydroxysuccinimide activation, epoxide activation, sulfhydrylactivation, and hydrazide activation. The resulting medium willgenerally be configured in the form of a column, and fluids containingligand are passed through the column one or more times to allow ligand(e.g., zsig32 polypeptide) to bind to the receptor/binding protein. Theligand is then eluted using changes in salt concentration, chaotropicagents (guanidine HCl), or pH to disrupt ligand-receptor binding.

[0125] An assay system that uses a ligand-binding receptor (or anantibody, one member of a complement/anti-complement pair) or a bindingfragment thereof, and a commercially available biosensor instrument(BIAcore™, Pharmacia Biosensor, Piscataway, N.J.) may be advantageouslyemployed. Such receptor, antibody, member of acomplement/anti-complement pair or fragment is immobilized onto thesurface of a receptor chip. Use of this instrument is disclosed byKarlsson, J. Immunol. Methods 145:229-40, 1991 and Cunningham and Wells,J. Mol. Biol. 234:554-63, 1993. A receptor, antibody, member or fragmentis covalently attached, using amine or sulfhydryl chemistry, to dextranfibers that are attached to gold film within the flow cell. A testsample is passed through the cell. If a ligand, epitope, or oppositemember of the complement/anti-complement pair is present in the sample,it will bind to the immobilized receptor, antibody or member,respectively, causing a change in the refractive index of the medium,which is detected as a change in surface plasmon resonance of the goldfilm. This system allows the determination of on- and off-rates, fromwhich binding affinity can be calculated, and assessment ofstoichiometry of binding.

[0126] Ligand-binding receptor polypeptides can also be used withinother assay systems known in the art. Such systems include Scatchardanalysis for determination of binding affinity (see Scatchard, Ann. NYAcad. Sci. 51: 660-72, 1949) and calorimetric assays (Cunningham et al.,Science 253:545-48, 1991; Cunningham et al., Science 245:821-25, 1991).

[0127] Zsig32 can be measured in vitro using cultured cells or in vivoby administering molecules of the claimed invention to the appropriateanimal model. For instance, zsig32 transfected (or co-transfected)expression host cells may be embedded in an alginate environment andinjected (implanted) into recipient animals. Alginate-poly-L-lysinemicroencapsulation, permselective membrane encapsulation and diffusionchambers have been described as a means to entrap transfected mammaliancells or primary mammalian cells. These types of non-immunogenic“encapsulations” or microenvironments permit the transfer of nutrientsinto the microenvironment, and also permit the diffusion of proteins andother macromolecules secreted or released by the captured cells acrossthe environmental barrier to the recipient animal. Most importantly, thecapsules or microenvironments mask and shield the foreign, embeddedcells from the recipient animal's immune response. Suchmicroenvironments can extend the life of the injected cells from a fewhours or days (naked cells) to several weeks (embedded cells).

[0128] Alginate threads provide a simple and quick means for generatingembedded cells. The materials needed to generate the alginate threadsare readily available and relatively inexpensive. Once made, thealginate threads are relatively strong and durable, both in vitro and,based on data obtained using the threads, in vivo. The alginate threadsare easily manipulable and the methodology is scalable for preparationof numerous threads. In an exemplary procedure, 3% alginate is preparedin sterile H₂O, and sterile filtered. Just prior to preparation ofalginate threads, the alginate solution is again filtered. Anapproximately 50% cell suspension (containing about 5×10⁵ to about 5×10⁷cells/ml) is mixed with the 3% alginate solution. One ml of thealginate/cell suspension is extruded into a 100 mM sterile filteredCaCl₂ solution over a time period of ^(˜)15 min forming a “thread”. Theextruded thread is then transferred into a solution of 50 mM CaCl₂, andthen into a solution of 25 mM CaCl₂. The thread is then rinsed withdeionized water before coating the thread by incubating in a 0.01solution of poly-L-lysine. Finally, the thread is rinsed with LactatedRinger's Solution and drawn from solution into a syringe barrel (withoutneedle attached). A large bore needle is then attached to the syringe,and the thread is intraperitoneally injected into a recipient in aminimal volume of the Lactated Ringer's Solution.

[0129] An alternative in vivo approach for assaying proteins of thepresent invention involves viral delivery systems. Exemplary viruses forthis purpose include adenovirus, herpesvirus, vaccinia virus andadeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus,is currently the best studied gene transfer vector for delivery ofheterologous nucleic acid (for a review, see T. C. Becker et al., Meth.Cell Biol. 43:161-89, 1994; and Douglas and Curiel, Science & Medicine4:44-53, 1997). The adenovirus system offers several advantages:adenovirus can (i) accommodate relatively large DNA inserts; (ii) begrown to high-titer; (iii) infect a broad range of mammalian cell types;and (iv) be used with a large number of available vectors containingdifferent promoters. Also, because adenoviruses are stable in thebloodstream, they can be administered by intravenous injection.

[0130] By deleting portions of the adenovirus genome, larger inserts (upto 7 kb) of heterologous DNA can be accommodated. These inserts can beincorporated into the viral DNA by direct ligation or by homologousrecombination with a co-transfected plasmid. In an exemplary system, theessential E1 gene has been deleted from the viral vector, and the viruswill not replicate unless the E1 gene is provided by the host cell (thehuman 293 cell line is exemplary). When intravenously administered tointact animals, adenovirus primarily targets the liver. If theadenoviral delivery system has an E1 gene deletion, the virus cannotreplicate in the host cells. However, the host's tissue (e.g., liver)will express and process (and, if a secretory signal sequence ispresent, secrete) the heterologous protein. Secreted proteins will enterthe circulation in the highly vascularized liver, and effects on theinfected animal can be determined.

[0131] The adenovirus system can also be used for protein production invitro. By culturing adenovirus-infected non-293 cells under conditionswhere the cells are not rapidly dividing, the cells can produce proteinsfor extended periods of time. For instance, BHK cells are grown toconfluence in cell factories, then exposed to the adenoviral vectorencoding the secreted protein of interest. The cells are then grownunder serum-free conditions, which allows infected cells to survive forseveral weeks without significant cell division. Alternatively,adenovirus vector infected 293S cells can be grown in suspension cultureat relatively high cell density to produce significant amounts ofprotein (see Garnier et al., Cytotechnol. 15:145-55, 1994). With eitherprotocol, an expressed, secreted heterologous protein can be repeatedlyisolated from the cell culture supernatant. Within the infected 293Scell production protocol, non-secreted proteins may also be effectivelyobtained. Zsig32 polypeptides can also be used to prepare antibodiesthat specifically bind to zsig32 epitopes, peptides or polypeptides.Antibodies generated from this immune response can be isolated andpurified as described herein. Methods for preparing and isolatingpolyclonal and monoclonal antibodies are well known in the art. See, forexample, Current Protocols in Immunology, Cooligan, et al. (eds.),National Institutes of Health, John Wiley and Sons, Inc., 1995; Sambrooket al., Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor, N.Y., 1989; and Hurrell, J. G. R., Ed., MonoclonalHybridoma Antibodies: Techniques and Applications, CRC Press, Inc., BocaRaton, Fla., 1982.

[0132] As would be evident to one of ordinary skill in the art,polyclonal antibodies can be generated from inoculating a variety ofwarm-blooded animals such as horses, cows, goats, sheep, dogs, chickens,rabbits, mice, hamsters, guinea pigs and rats as well as transgenicanimals such as transgenic sheep, cows, goats or pigs. Antibodies mayalso be expressed in yeast and fungi in modified forms as well as inmammalian and insect cells. The zsig32 polypeptide or a fragment thereofserves as an antigen (immunogen) to inoculate an animal or elicit animmune response. Suitable antigens would include the zsig32 polypeptideencoded by SEQ ID NO:2 from amino acid residue 21-175 of SEQ ID NO:2, ora contiguous 9-175 amino acid residue fragment thereof. Theimmunogenicity of a zsig32 polypeptide may be increased through the useof an adjuvant, such as alum (aluminum hydroxide) or Freund's completeor incomplete adjuvant. Polypeptides useful for immunization alsoinclude fusion polypeptides, such as fusions of zsig32 or a portionthereof with an immunoglobulin polypeptide or with maltose bindingprotein. The polypeptide immunogen may be a full-length molecule or aportion thereof. If the polypeptide portion is “hapten-like”, suchportion may be advantageously joined or linked to a macromolecularcarrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin(BSA) or tetanus toxoid) for immunization.

[0133] As used herein, the term “antibodies” includes polyclonalantibodies, affinity-purified polyclonal antibodies, monoclonalantibodies, and antigen-binding fragments, such as F(ab′)₂ and Fabproteolytic fragments. Genetically engineered intact antibodies orfragments, such as chimeric antibodies, Fv fragments, single chainantibodies and the like, as well as synthetic antigen-binding peptidesand polypeptides, are also included. Non-human antibodies may behumanized by grafting non-human CDRs onto human framework and constantregions, or by incorporating the entire non-human variable domains(optionally “cloaking” them with a human-like surface by replacement ofexposed residues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced. Human antibodies can also be madein mice having a humanized humoral immune system (Mendez et al., Nat.Genet. 14:146-56, 1997).

[0134] Alternative techniques for generating or selecting antibodiesuseful herein include in vitro exposure of lymphocytes to zsig32 proteinor peptide, and selection of antibody display libraries, in phage orsimilar vectors (for instance, through use of immobilized or labeledzsig32 protein or peptide). Mutagenesis methods discussed herein, inparticular domain shuffling, can be used to generate and matureantibodies.

[0135] The antibodies of the current invention, or fragments thereof,can be used to direct molecules to a specific target. For example, asT-bodies, chimeric receptors combining antibody recognition with T celleffector function, (Eshhar et al., Springer Semin Immunopathol.18:199-209, 1996; Eshhar, Cancer Immunol. Immunother. 45:131-6, 1997).Intrabodies, engineered single-chain antibodies expressed inside thecell and having high affinity and specificity for intracellular targets.Such molecules have use in gene therapy and treatment of infectiousdiseases (Marasco, Immunotechnology 1:1-19, 1995; Marasco et al., GeneTher. 4:11-5, 1997; Rondon and Marasco, Annu. Rev. Microbiol. 51:257-83,1997 and Mhashilkar et al., J. Virol. 71:6486-94, 1997). Diabodies,bispecific non-covalent dimers of scFv antibodies useful forimmunodiagnosis and therapeutically. In addition they can be constructedin bacteria (Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48,1993).

[0136] Antibodies herein specifically bind if they bind to a zsig32polypeptide, peptide or epitope with a binding affinity (K_(a)) of 10⁶M⁻¹ or greater, preferably 10⁷ M⁻¹ or greater, more preferably 10⁸ M⁻¹or greater, and most preferably 10⁹ M⁻¹ or greater. The binding affinityof an antibody can be readily determined by one of ordinary skill in theart, for example, by Scatchard analysis (Scatchard, ibid.).

[0137] Genes encoding polypeptides having potential zsig32 polypeptidebinding domains, “binding proteins”, can be obtained by screening randomor directed peptide libraries displayed on phage (phage display) or onbacteria, such as E. coli. Nucleotide sequences encoding thepolypeptides can be obtained in a number of ways, such as through randommutagenesis and random polynucleotide synthesis. Alternatively,constrained phage display libraries can also be produced. These peptidedisplay libraries can be used to screen for peptides which interact witha known target which can be a protein or polypeptide, such as a ligandor receptor, a biological or synthetic macromolecule, or organic orinorganic substances. Techniques for creating and screening such peptidedisplay libraries are known in the art (Ladner et al., U.S. Pat. No.5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al., U.S.Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No. 5,571,698) andpeptide display libraries and kits for screening such libraries areavailable commercially, for instance from Clontech (Palo Alto, Calif.),Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly,Mass.) and Pharmacia LKB Biotechnology Inc. (Piscataway, N.J.). Peptidedisplay libraries can be screened using the zsig32 sequences disclosedherein to identify proteins which bind to zsig32. These “bindingproteins” which interact with zsig32 polypeptides can be usedessentially like an antibody, for tagging cells; for isolating homologpolypeptides by affinity purification; directly or indirectly conjugatedto drugs, toxins, radionuclides and the like. These binding proteins canalso be used in analytical methods such as for screening expressionlibraries and neutralizing activity. The binding proteins can also beused for diagnostic assays for determining circulating levels ofpolypeptides; for detecting or quantitating soluble polypeptides asmarker of underlying pathology or disease. To increase the half-life ofthese binding proteins, they can be conjugated. Their biologicalproperties may be modified by dimerizing or multimerizing for use asagonists or antagonists.

[0138] A variety of assays known to those skilled in the art can beutilized to detect antibodies and/or binding proteins which specificallybind to zsig32 proteins or peptides. Exemplary assays are described indetail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), ColdSpring Harbor Laboratory Press, 1988. Representative examples of suchassays include: concurrent immunoelectrophoresis, radioimmunoassay,radioimmuno-precipitation, enzyme-linked immunosorbent assay (ELISA),dot blot or Western blot assay, inhibition or competition assay, andsandwich assay. In addition, antibodies can be screened for binding towild-type versus mutant zsig32 protein or polypeptide.

[0139] Antibodies and binding proteins to zsig32 may be used for taggingcells that express zsig32; for isolating zsig32 by affinitypurification; for diagnostic assays for determining circulating levelsof zsig32 polypeptides; for detecting or quantitating soluble zsig32 asmarker of underlying pathology or disease; in analytical methodsemploying FACS; for screening expression libraries; for generatinganti-idiotypic antibodies; and as neutralizing antibodies or asantagonists to block zsig32 polypeptide adhesion modulating oranti-microbial or like activity in vitro and in vivo. Suitable directtags or labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent markers, chemiluminescent markers, magneticparticles and the like; indirect tags or labels may feature use ofbiotin-avidin or other complement/anti-complement pairs asintermediates. Antibodies herein may also be directly or indirectlyconjugated to drugs, toxins, radionuclides and the like, and theseconjugates used for in vivo diagnostic or therapeutic applications.Moreover, antibodies to zsig32 or fragments thereof may be used in vitroto detect denatured zsig32 or fragments thereof in assays, for example,Western Blots or other assays known in the art.

[0140] In another embodiment, polypeptide-toxin fusion proteins orantibody-toxin fusion proteins can be used for targeted cell or tissueinhibition or ablation (for instance, to treat cancer cells or tissues).Alternatively, if the polypeptide has multiple functional domains (i.e.,an activation domain or a ligand binding domain, plus a targetingdomain), a fusion protein including only the targeting domain may besuitable for directing a detectable molecule, a cytotoxic molecule or acomplementary molecule to a cell or tissue type of interest. Ininstances where the domain only fusion protein includes a complementarymolecule, the anti-complementary molecule can be conjugated to adetectable or cytotoxic molecule. Such domain-complementary moleculefusion proteins thus represent a generic targeting vehicle forcell/tissue-specific delivery of genericanti-complementary-detectable/cytotoxic molecule conjugates. Thebioactive polypeptide or antibody conjugates described herein can bedelivered intravenously, intraarterially, intraductally with DMSO,intramuscularly, subcutaneously, intraperitoneally, also by transdermalmethods, by electro-transfer, orally or via inhalant. Molecules of thepresent invention can be used to identify and isolate receptors involvedin salivary gland function or saliva composition. For example, proteinsand peptides of the present invention can be immobilized on a column andmembrane preparations run over the column (Immobilized Affinity LigandTechniques, Hermanson et al., eds., Academic Press, San Diego, Calif.,1992, pp.195-202). Proteins and peptides can also be radiolabeled(Methods in Enzymol., vol. 182, “Guide to Protein Purification”, M.Deutscher, ed., Acad. Press, San Diego, 1990, 721-737) or photoaffinitylabeled (Brunner et al., Ann. Rev. Biochem. 62:483-514, 1993 and Fedanet al., Biochem. Pharmacol. 33:1167-1180, 1984) and specificcell-surface proteins can be identified.

[0141] For pharmaceutical use, the proteins of the present invention areformulated for parenteral, particularly intravenous or subcutaneous,delivery according to conventional methods. Intravenous administrationwill be by bolus injection or infusion over a typical period of one toseveral hours. In general, pharmaceutical formulations will include azsig32 protein in combination with a pharmaceutically acceptablevehicle, such as saline, buffered saline, 5% dextrose in water or thelike. Formulations may further include one or more excipients,preservatives, solubilizers, buffering agents, albumin to preventprotein loss on vial surfaces, etc. Methods of formulation are wellknown in the art and are disclosed, for example, in Remington: TheScience and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co.,Easton, Pa., 19th ed., 1995. Therapeutic doses will generally determinedby the clinician according to accepted standards, taking into accountthe nature and severity of the condition to be treated, patient traits,etc. Determination of dose is within the level of ordinary skill in theart. The proteins may be administered for acute treatment, over one weekor less, often over a period of one to three days or may be used inchronic treatment, over several months or years.

[0142] The invention is further illustrated by the followingnon-limiting examples.

EXAMPLES Example 1 Extension of EST Sequence

[0143] The novel zsig32 polypeptide-encoding polynucleotides of thepresent invention were initially identified by querying an EST databasefor secretory signal sequences characterized by an upstream methioninestart site, a hydrophobic region of approximately 13 amino acids and acleavage site (SEQ ID NO: 6, wherein cleavage occurs between the alanineand glycine amino acid residues) in an effort to select for secretedproteins. Polypeptides corresponding to ESTs meeting those searchcriteria were compared to known sequences to identify secreted proteinshaving homology to known ligands. A single EST sequence was discoveredand predicted to be related to a mouse ventral prostate spermine-bindingprotein (SPBP). See, for example, Mills et al. cited above. To identifythe corresponding cDNA, a clone considered likely to contain the entirecoding region was used for sequencing. Using an Invitrogen S.N.A.P.™Miniprep kit (Invitrogen, Corp., San Diego, Calif.) according tomanufacturer's instructions a 5 ml overnight culture in LB+50 μg/mlampicillin was prepared. The template was sequenced on an ABIPRISM™model 377 DNA sequencer (Perkin-Elmer Cetus, Norwalk, Ct.) using the ABIPRISM™ Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-ElmerCorp.) according to manufacturer's instructions. Oligonucleotides ZC694(SEQ ID NO: 8), ZC695 (SEQ ID NO: 9) to the T7 and SP6 promoters on theclone-containing vector were used as sequencing primers.Oligonucleotides ZC13183 (SEQ ID NO: 10), ZC13187 (SEQ ID NO: 11) wereused to complete the sequence from the clone. Sequencing reactions werecarried out in a Hybaid OmniGene Temperature Cycling System (NationalLabnet Co., Woodbridge, N.Y.). SEQUENCHER™ 3.0 sequence analysissoftware (Gene Codes Corporation, Ann Arbor, Mich.) was used for dataanalysis. The resulting 853 bp sequence is disclosed in SEQ ID NO: 1.

Example 2 Tissue Distribution

[0144] Northerns were performed using Human Multiple Tissue Blots fromClontech (Palo Alto, Calif.). A 40 bp DNA probe (ZC12493; SEQ ID NO: 7)to the 5′ end of the oligonucleotide sequence of the mature polypeptideshown in SEQ ID NO: 1 was radioactively labeled with ³²P using T4polynucleotide kinase and forward reaction buffer (GIBCO BRL,Gaithersburg, Md.) according to the manufacturer's specifications. Theprobe was purified using a NUCTRAP push column (Stratagene CloningSystems, La Jolla, Calif.). EXPRESSHYB (Clontech, Palo Alto, Calif.)solution was used for prehybridization and as a hybridizing solution forthe Northern blots. Hybridization took place overnight at 42° C, and theblots were then washed in 2×SSC and 0.05% SDS at RT, followed by a washin 1×SSC and 0.1% SDS at 71° C. One transcript size was observed atapproximately 650 bp. Signal intensity was highest for prostate, stomachand trachea, with relatively less intense signals in spleen and colon.

[0145] A RNA Master Dot Blot (Clontech) that contained RNAs from varioustissues that were normalized to 8 housekeeping genes was also probedwith the 40 bp DNA probe (SEQ ID NO: 7). The blot was prehybridized andthen hybridized overnight with 10⁶ cpm/ml of the probe at 42° C.,according to the manufacturer's specifications. The blot was washed with2×SSC and 0.05% SDS at RT, followed by a wash in 0.1×SSC and 0.1% SDS at71° C. After a 48 hour exposure, highest expression was seen in thesalivary gland, with much weaker signals in trachea and still weakersignals in prostate. Note that in prostate and trachea, a 2 kb band wasalso observed.

Example 3 Chromosomal Assignment and Placement of zsig32

[0146] Zsig32 was mapped to chromosome 16 using the commerciallyavailable GeneBridge 4 Radiation Hybrid Panel (Research Genetics, Inc.,Huntsville, Ala.). The GeneBridge 4 Radiation Hybrid Panel containsPCRable DNAs from each of 93 radiation hybrid clones, plus two controlDNAs (the HFL donor and the A23 recipient). A publicly available wwwserver (http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl) allowsmapping relative to the Whitehead Institute/MIT Center for GenomeResearch's radiation hybrid map of the human genome (the “WICGR”radiation hybrid map) which was constructed with the GeneBridge 4Radiation Hybrid Panel.

[0147] For the mapping of zsig32 with the GeneBridge 4 RH Panel, 20 Alreactions were set up in a 96-well microtiter plate (Stratagene) andused in a RoboCycler Gradient 96 thermal cycler (Stratagene). Each ofthe 95 PCR reactions consisted of 2 μl 10×KlenTaq PCR reaction buffer(Clontech), 1.6 μl dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City,Calif.), 1 μl sense primer, ZC 13,703 (SEQ ID NO:21), 1 μl antisenseprimer, ZC 13,704 (SEQ ID NO:22), 2 μl RediLoad (Research Genetics,Inc.), 0.4 μl 50×Advantage KlenTaq Polymerase Mix (ClontechLaboratories, Inc.), 25 ng of DNA from an individual hybrid clone orcontrol and ddH₂O for a total volume of 20 μl. The reactions wereoverlaid with an equal amount of mineral oil and sealed. The PCR cyclerconditions were as follows: an initial 1 cycle 5 minute denaturation at95° C., 35 cycles of a 1 minute denaturation at 95° C., 1 minuteannealing at 60° C. and 1.5 minute extension at 72° C., followed by afinal 1 cycle extension of 7 minutes at 72° C. The reactions wereseparated by electrophoresis on a 2% agarose gel (Life Technologies,Gaithersburg, Md.).

[0148] The results showed that Zsig32 maps 7.47 cR_(—)3000 from theframework marker WI-7742 on the WICGR chromosome 16 radiation hybridmap. Proximal and distal framework markers were WI-7742 (D16S2960) andWI-3061 (D16S2965), respectively. The use of surrounding markerspositions Zsig32 in the 16p13.3 region on the integrated LDB chromosome16 map (The Genetic Location Database, University of Southhampton, wwwserver: http://cedar.genetics.soton.ac.uk/public_html/).

Example 4 Creation of Mammalian Expression Vectors Zsig32NF/pZP9,Zsig32CF/pZP9 and Zsig32/pZP9

[0149] Three expression vectors were prepared for the zsig32polypeptide, zSIG32CF/pZP9 and zSIG32NF/pZP9, wherein the constructs aredesigned to express a zsig25 polypeptide with a C- or N-terminal FLAGtag (SEQ ID NO: 16) and zSIG32/pZP9 expressing untagged zsig32polypeptides.

[0150] ZSIG32/pZP9

[0151] A approximately 800 bp restriction digest fragment of ZSIG-32 DNAwas derived from the clone described in Example 1 above. Ten microlitersof the clone was digested with 1.5 μl each of the restriction enzymesEco RI and Not I. The resultant ligation fragment was then run on a 0.8%LMP agarose gel (Seaplaque GTG) with 0.5×TBE buffer. A band of thepredicted size was excised and the DNA was purified from the gel with aQIAQUICK® column (Qiagen) according the manufacturer's instructions.

[0152] The excised, restriction digested zsig32 DNA was subcloned intoplasmid pZP9 which had been cut with Eco RI and Not I. Plasmid CF/pZP9(deposited at the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md.) is a mammalian expression vector containing anexpression cassette having the mouse metallothionein-1 promoter,multiple restriction sites for insertion of coding sequences, a stopcodon and a human growth hormone terminator. The plasmid also has an E.coli origin of replication, a mammalian selectable marker expressionunit having an SV40 promoter, enhancer and origin of replication, a DHFRgene and the SV40 terminator.

[0153] zSIG25CF/pZP9

[0154] A 553 bp PCR generated ZSIG-32 DNA fragment was created usingZC13465 (SEQ ID NO:23) and ZC13447 (SEQ ID NO:26) as PCR primers and thetemplate described in Example 1 above. The PCR reaction was incubated at94° C. for 5 minutes, and then run for 10 cycles of 30 seconds at 94° C.and 2 minutes at 72° C., followed by 15 cycles at 94° C. for 30 secondsand 65° C. for 2 minutes. The resultant PCR product was then run on a0.9% GTG/TBE agarose gel with lx TBE buffer. A band of the predictedsize was excised and the DNA was purified from the gel with a QIAQUIC™column (Qiagen) according the manufacturer's instructions. The DNA wasdigested with the restriction enzymes BAM HI (Boehringer Mannheim) andEco RI (Gibco BRL), followed by phenol/chloroform/isoamyl alcoholextraction and ETOH/glycogen precipitated.

[0155] The excised, restriction digested zsig32 DNA was subcloned intoplasmid CF/pZP9 which had been cut with Eco RI and Bam HI. ThezSIG32/CFpZP9 expression vector uses the native zSIG32 signal peptide,and the FLAG epitope (SEQ ID NO:16) is attached at the C-terminus as apurification aid. Plasmid CF/pZP9 (deposited at the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md.) is a mammalianexpression vector containing an expression cassette having the mousemetallothionein-1 promoter, multiple restriction sites for insertion ofcoding sequences, a sequence encoding the FLAG tag (SEQ ID NO:16), astop codon and a human growth hormone terminator. The plasmid also hasan E. coli origin of replication, a mammalian selectable markerexpression unit having an SV40 promoter, enhancer and origin ofreplication, a DHFR gene and the SV40 terminator.

[0156] ZSIG25NF/pZP9

[0157] A 490 bp PCR generated zSIG32/NF DNA fragment was created inaccordance with the procedure set forth above using Z13448 (SEQ IDNO:25) and ZC13449 (SEQ ID NO:26) as PCR primers. The purified PCRfragment was digested with the restriction enzymes Bam HI (BoehringerMannheim) and Xho I (Gibco BRL), followed by phenol/chloroform/isoamylalcohol extraction and ETOH/glycogen precipitation.

[0158] The excised and restriction digested zSIG32 DNA was subclonedinto plasmid NF/pZP9 which had been cut with Bam HI and Xba I. ThezSIG32/NFpZP9 expression vector incorporates the TPA leader and attachesthe FLAG tag (SEQ ID NO:16) to the N-terminal of the zsig25polypeptide-encoding polynucleotide sequence. Plasmid NF/pZP9 (depositedat the American Type Culture Collection, 12301 Parklawn Drive,Rockville, Md.) is a mammalian expression vector containing anexpression cassette having the mouse metallothionein-1 promoter, a TPAleader peptide followed by the sequence encoding the FLAG tag (SEQ IDNO:16), multiple restriction sites for insertion of coding sequences,and a human growth hormone terminator. The plasmid also contains an E.coli origin of replication, a mammalian selectable marker expressionunit having an SV40 promoter, enhancer and origin of replication, a DHFRgene and the SV40 terminator.

[0159] For the untagged zsig32 construct 100 ng of the zsig32 insert and90 ng of the Eco RI/Not I digested pZP9 vector were ligated as describedfor the tagged constructs. For the N- and C-tagged constructs, 10 ng ofthe restriction digested inserts and 20 ng of the corresponding vectorswere ligated at room temperature for 4 hours. One microliter of eachligation reaction was independently electroporated into DH10B competentcells (GIBCO BRL, Gaithersburg, Md.) according to manufacturer'sdirection and plated onto LB plates containing 50 mg/ml ampicillin, andincubated overnight. Colonies were screened by PCR as described above.For zsig32/pZP9 screens the primers were ZC6583 (SEQ ID NO:27) andZC5020 (SEQ ID NO:28), for zSIG32CF/pZP9 screens the primers were,ZC13465 (SEQ ID NO:23) and ZC13447 (SEQ ID NO:24) and for zSIG32NF/pZP9screens the primers were ZC13448 (SEQ ID NO:25) and ZC13449 (SEQ IDNO:26). The insert sequence of positive clones, 1000 bp for zsig32untagged, 490 bp fragment for zSIG32NF and a 553 bp fragment forzSIG32/CF were verified by sequence analysis. A large scale plasmidpreparation was done using a QIAGEN Maxi prep kit (Qiagen) according tomanufacturer's instructions.

Example 5 Mammalian Expression of zsig32

[0160] BHK 570 cells (ATCC NO: CRL-10314) were plated in 10 cm tissueculture dishes and allowed to grow to approximately 50 to 70% confluencyovernight at 37° C., 5% CO₂, in DMEM/FBS media (DMEM, Gibco/BRL HighGlucose, (Gibco BRL, Gaithersburg, Md.), 5% fetal bovine serum (Hyclone,Logan, Utah), 1 μL-glutamine (JRH Biosciences, Lenexa, Kans.), 1 μMsodium pyruvate (Gibco BRL)). The cells were then transfected with theplasmid zsig32NF/pZP9 (N-terminal FLAG tag), zsig32CF/pZP9 (C-terminalFLAG tag), or zsig32/pZP9 (untagged), using Lipofectamine M (Gibco BRL),in serum free (SF) media formulation (DMEM, 10 mg/ml transferrin, 5mg/ml insulin, 2 mg/ml fetuin, 1% L-glutamine and 1 sodium pyruvate).Sixteen micrograms of zsig32NF/pZP9 and 16 μg of zsig32CF/pZP9 wereseparately diluted into 15 ml tubes to a total final volume of 640 μlwith SF media. In separate tubes, 35 μl of Lipofectamine™ (Gibco BRL)was mixed with 605 μl of SF medium. The Lipofectamine™ mix was added tothe DNA mix and allowed to incubate approximately 30 minutes at roomtemperature. Five milliliters of SF media was added to theDNA:Lipofectamine™ mixture. Three plates of cells were rinsed once with5 ml of SF media, aspirated, and the DNA:Lipofectamine™ mixture wasadded. The cells were incubated at 37° C. for five hours, then 6.4 ml ofDMEM/10% FBS, 1% PSN media was added to each plate. The plates wereincubated at 37° C. overnight and the TM DNA:Lipofectamine mixture wasreplaced with fresh FBS/DMEM media the next day. On day 2post-transfection, the cells were split into the selection media(DMEM/FBS media from above with the addition of 1 μM methotrexate (SigmaChemical Co., St. Louis, Mo.)) in 150 mm plates at 1:10, 1:20 and 1:50.The cells were refed at day 5 post-transfection with fresh selectionmedia. Approximately 12 days post-transfection, two 150 mm culturedishes of methotrexate resistant colonies from each transfection weretrypsinized and the cells were pooled and plated into a T-162 flask andtransferred to large scale culture.

Example 6 Large Scale Mammalian Expression of zsig32

[0161] One T-162 flask, containing confluent cells expressing zsig32/NFand one flask containing zsig32/CF expressing cells, obtained from theexpression procedure described above, were expanded into five T-162flasks. One of the five resulting flasks was used to freeze down fourcryovials, and the other four flasks were used to generate a Nunc cellfactory.

[0162] The cells from the four T-165 flasks of zsig32/NF and zsig32/CFwere combined and used to seed two Nunc cell factories (10 layers,commercially available from VWR). Briefly, the cells from the T-162flasks described above were detached using trypsin, pooled, and added to1.5 liters ESTEP1 media (668.7g/50L DMEM (Gibco), 5.5 g/50L pyruvicacid, sodium salt 96% (Mallinckrodt), 185.0 g/SOL NaHCO₃ (Mallinkrodt),5.0 mg/ml and 25 ml/50L insulin (JRH Biosciences), 10.0 mg/ml and 25ml/50L transferrin (JRH Biosciences), 2.5L/50L fetal bovine serum(characterized) (Hyclone), 1 μM MTX, with pH adjusted to 7.05+/−0.05)prewarmed to 37° C. The media containing the cells was then poured intothe Nunc cell factories via a funnel. The cell factories were placed ina 37° C./5.0% CO2 incubator.

[0163] At 80-100% confluence, a visual contamination test (phenol redcolor change) was performed on the Nunc cell factories. Since nocontamination was observed, supernatant from the confluent factories waspoured into a small harvest container, sampled and discarded. Theadherent cells were then washed once with 400 ml PBS. To detach thecells from the factories, 100 mls of trypsin was added to each andremoved and the cells were then incubated for 5 to 10 minutes in theresidual trypsin. The cells were collected following two, 200 ml washesof ESTEP1 media. To each of ten ESTEP1 media-containing bottles (1.5liters each, at 37° C.) was added 40 mls of collected cells. One 1.5liter bottle was then used to fill one Nunc factory. Each cell factorywas placed in a 37° C./5.0% CO₂ incubator.

[0164] At 80-90% confluence, a visual contamination test (phenol redcolor change) was performed on the Nunc cell factories. Since nocontamination was observed, supernatant from the confluent factories waspoured into a small harvest container, sampled and discarded. Cells werethen washed once with 400 ml PBS. 1.5 liters of ESTEP2 media (668.7g/50LDMEM (Gibco), 5.5 g/SOL pyruvic acid, sodium salt 96% (Mallinckrodt),185.0 g/50L NaHCO₃ (Mallinkrodt), 5.0 mg/ml, 25 ml/50L insulin, 10.0mg/ml and 25 ml/50L transferrin) was added to each Nunc cell factory.The cell factories were incubated at 37° C./5.0% CO₂.

[0165] At approximately 48 hours a visual contamination test (phenol redcolor change) was performed on the Nunc cell factories. Supernatant fromeach factory was poured into small harvest containers. A total of 13.5liters was collected from all 10 factories. Fresh serum-free media (1.5liters) was poured into each Nunc cell factory, and the factories wereincubated at 37° C./5.0% CO₂. One ml of supernatant harvest wastransferred to a microscope slide, and subjected to microscopic analysisfor contamination. The contents of the small harvest containers for eachfactory were pooled and immediately filtered. A second harvest was thenperformed, substantially as described above at 44 hours (13.5 L wereobtained) and the cell factories were discarded thereafter. Anaseptically assembled filter train apparatus was used for asepticfiltration of the harvest supernatant (conditioned media). Assembly wasa follows: tubing was wire-tied to an Opti-Cap filter (Millipore Corp.,Bedford, Mass.) and a Gelman Supercap 50 filter (Gelman Sciences, AnnArbor, Mich.). The Supercap 50 filter was also attached to a sterilecapped container located in a hood; tubing located upstream of theMillipore Opti-cap filter was inserted into a peristaltic pump; and thefree end of the tubing was placed in the large harvest container. Theperistaltic pump was run between 200 and 300 rpm, until all of theconditioned media passed through the 0.22 μm final filter into a sterilecollection container. The filtrate was placed in a 4° C. cold roompending purification. The media was concentrated 10×with a Millipore 5kDA cut off concentrator (Millipore Corp., Bedford, Mass.) according tomanufacturer's direction and subjected to Western Blot analysis using ananti-FLAG tag antibody (Kodak).

Example 7 Purification of Mammalian Expressed FLAG-tagged zsig32Polypeptides

[0166] Unless otherwise noted, all operations were carried out at 4° C.The following procedure was used to purify zsig32 constructs having anN-terminal or C-terminal flag tag. Protein was purified from the culturemedium of a mixture of baby hamster kidney cell (BHK) clones thatproduced the N- or C-terminal tagged protein (zsig32NF and zsig32CF). Atotal of 25 liters of conditioned media from BHK cells was sequentiallysterile filtered through a 4 inch, 0.2 mM Millipore (Bedford, Mass.)OptiCap capsule filter and a 0.2 mM Gelman (Ann Arbor, Mich.) Supercap50. The material was then concentrated to about 1.3 liters using anAmicon (Beverly, Mass.) DC 10L concentrator fitted with an A/G Tech(Needham, Mass.) hollow fiber cartridge with a 15 sq. ft. 3000 kDacutoff membrane. The concentrated material was again sterile-filteredwith the Gelman filter as described above. A 25.0 ml sample of anti-FlagSepharose (Eastman Kodak, Rochester, N.Y.) was added to the sample forbatch adsorption and the mixture was gently agitated on a Wheaton(Millville, N.J.) roller culture apparatus for 18.0 h at 4° C.

[0167] The mixture was then poured into a 5.0×20.0 cm Econo-Column(Bio-Rad, Laboratories, Hercules, Calif.) and the gel was washed with 30column volumes of phosphate buffered saline (PBS). The unretainedflow-through fraction was discarded. Once the absorbance of the effluentat 280 nM was less than 0.05, flow through the column was reduced tozero and the anti-Flag Sepharose gel was washed with 2.0 column volumesof PBS containing 0.2 mg/ml of Flag peptide,N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C (SEQ ID NO:16) (Eastman Kodak).After 1.0 hour at 4° C., flow was resumed and the eluted protein wascollected. This fraction is referred to as the peptide elution. Theanti-Flag Sepharose gel was washed with 2.0 column volumes of 0.1Mglycine, pH 2.5, and the glycine wash was collected separately. The pHof the glycine-eluted fraction was adjusted to 7.0 by the addition of asmall volume of 10×PBS and stored at 4° C. for future analysis ifneeded.

[0168] The peptide elution was concentrated to 5.0 ml using a 5,000molecular weight cutoff membrane concentrator (Millipore, Bedford,Mass.) according to the manufacturer's instructions. The concentratedpeptide elution was then separated from free peptide by chromatographyon a 1.5×50 cm Sephadex G-50 (Pharmacia, Piscataway, N.J.) columnequilibrated in PBS at a flow rate of 1.0 ml/min using a BioCad SprintHPLC system (PerSeptive BioSystems, Framingham, Mass.). Two-ml fractionswere collected and the absorbance at 280 nM was monitored. The firstpeak of material absorbing at 280 nM and eluting near the void volume ofthe column was collected.

[0169] By SDS-PAGE and Western analysis with anti-Flag M2 antibodies(Kodak) the purified zsig32 NF/CF preparation was composed of one majorCoomassie Blue-stained band of apparent molecular weight 25,000. Twominor bands of apparent molecular weights 22,000 and 27,000, were alsoobserved on the Coomassie Blue-stained gel. All three bands showedcross-reactivity with the anti-Flag M2 antibody on Western blots.Migration of the proteins on SDS-PAGE gels and Western blots was notchanged by reducing agents.

[0170] The protein concentration of the purified proteins (0.67 mg/ml)was determined by BCA analysis (Pierce, Rockford, Ill.) and the materialwas aliquoted, and stored at −80° C.

Example 8 Creation of Baculovirus Expression Vectors pFSG32, pFSGE32,pSSGE32 and pLSGE32

[0171] Four expression vectors were prepared to express zsig32polypeptides in insect cells: pFSG32, designed to express an untaggedzsig32 polypeptide; and pFSGE32, pSSGE32 and pLSGE32, designed toexpress a zsig32 polypeptide with a C-terminal Glu-Glu tag (SEQ IDNO:12).

[0172] pFSG32

[0173] A 559 bp PCR generated zsig32 DNA fragment was created usingZC13404 (SEQ ID NO:13) and ZC13409 (SEQ ID NO:14) as PCR primers andzsig32/pZP9, described above, as a template. The PCR reaction wasincubated at 94° C. for 2 minutes, followed by 30 cycles of 45 secondsat 94° C., 1 minute at 60° C. and 72° C. for 1 minute with a 1second/cycle segment extension. The resultant PCR product was then runon a 3% gel (2% NuSieve/1% BRL agarose). The 559 bp fragment wascaptured by diluting 15 fold with 0.1 mM EDTA pH 8.0 and then ligatedinto the vector pCR2.1 (TA Cloning Kit, Invitrogen Inc., San Diego,Calif.) according to manufacturer's instructions. The resultant cloneswere screened for the proper insert orientation and sequenced to confirmidentity. The resulting clone, designated pSG32a, was digested with BamHI and Asp718 and the digest run on a 1% SeaPlaque/1%l NuSieve agarosegel. The band was excised, diluted to 0.5% agarose with 2 mM MgCl₂,melted at 65° C. and ligated into a Bam HI/Asp718 digested baculovirusexpression vector, pFastBac1 (Bac-to-Bac™ System, GIBCO-BRL,Gaithersburg, Md.). Forty four nanograms of the restriction digestedzsig32 insert and 126 ng of the corresponding vector were ligatedovernight. The ligation mix was diluted 3 fold in TE (10 mM Tris-HCl, pH7.5 and 1 mM EDTA) and 4 fmol of the diluted ligation mix wastransformed into DH5α Library Efficiency competent cells (LifeTechnologies) according to manufacturer's direction by heat shock for 45seconds in a 42° C. waterbath. The ligated DNA was diluted in 450 μl SOCmedia (2% Bacto Tryptone, 0.5% Bacto Yeast Extract, 10 ml 1M NaCl, 1.5mM KCl, 10 mM MgCl₂, 10 mM MgSO₄ and 20 mM glucose) and plated onto LBplates containing 100 μg/ml ampicillin. The plates were incubatedovernight at 37° C. Plasmid DNA was prepared using the QiaVac Miniprep8system (Qiagen) according the manufacturer's directions. The clones werescreened by restriction digest with Hind III/BspE1.

[0174] pFSGE32

[0175] A zsig32 fragment having a C-terminal Glu-Glu tag (SEQ ID NO:12)was generated by PCR as described above using oligonucleotide primersZC13404 (SEQ ID NO:13) and ZC13407 (SEQ ID NO:15). A fragment of theexpected size, 580 bp, was detected by gel electrophoresis. The DNAfragment was digested with the restriction enzymes Bam HI and Asp718 andthe resulting 565 bp zsig32 restriction fragment was ligated into a BamHI/Asp718 digested pFastBac1 vector and transformed into DH10α cells asdescribed above.

[0176] pSSGE32 and pLSGE32

[0177] A 580 bp zsig32 fragment having a C-terminal Glu-Glu tag (SEQ IDNO:12) was generated by PCR as described above. The fragment wasvisualized by gel electrophoresis, restriction digested and ligated intothe expression vectors, pFBPL2 and pFBPS2, as described above. Thevectors, pFBPL2 and pFBPS2, were derived from a modified pFastBacl™vector. The polyhedrin promoter was removed and substituted with a short(FBPS) or long (FBPL) version of the baculovirus basic protein promoteras is known in the art (Hill-Perkins and Possee, ibid.; Bonning et al.,ibid.; and, Chazenbalk and Rapoport, ibid.).

[0178] One microliter of each of the above constructs was used toindependently transform 20 μl DH10Bac Max Efficiency competent cells(GIBCO-BRL, Gaithersburg, Md.) according to manufacturer's instruction,by heat shock at 42° C. for 45 seconds. The transformants were thendiluted in an appropriate volume of SOC media and plated on to LuriaAgar plates containing 50 μg/ml kanamycin, 7 μg/ml gentamicin, 10 μg/mltetracycline, IPTG and Bluo Gal. The cells were incubated for 48 hoursat 37° C. A color selection was used to identify those cells havingvirus that had incorporated into the plasmid (referred to as a“bacmid”). Those colonies, which were white in color, were picked foranalysis. Bacmid DNA was isolated from positive colonies and screenedfor the correct insert using PCR oligonucleotide primers ZC976 (SEQ IDNO:17) and ZC447 (SEQ ID NO:18) were used and those having the correctinsert were used to transfect Spodoptera frugiperda (Sf9) cells.

[0179] Sf9 cells were seeded at 5×10⁶ cells per 35 mm plate and allowedto attach for 1 hour at 27° C. Five microliters of bacmid DNA wasdiluted with 100 μl Sf-900 II SFM. Six to 10 μl of CellFECTIN Reagent(Life Technologies) was diluted with 100 μl Sf-900 II SMF. The bacmidDNA and lipid solutions were gently mixed and incubated 30-45 minutes atroom temperature. The media from one plate of cells were aspirated, andthe lipid-DNA mixture to which 0.8 ml of Sf-900 II SFM was added. Thecells were incubated at 27° C. for 4-5 hours, then 2 ml of Sf-900 IImedia containing penicillin/streptomycin was added to each plate. Theplates were incubated at 27° C., 90% humidity, for 72 hours after whichthe virus was harvested.

[0180] Primary Amplification

[0181] Sf9 cells were grown in 50 ml Sf-900 II SFM in a 50 ml shakeflask to an approximate density of 0.04-0.50×10⁶ cells/ml. They werethen transfected with 50-1000 μl of the virus stock from above andincubated at 27° C. for 3-5 days after which time the virus washarvested, titer 0.53×10⁸ pfu/ml. To scale up, 1.5×10⁶ SF9 cells/ml wereadded to five liters of SF 900 II SFM and grown for 91 hours. The cellswere then transfected with the harvested virus (MOI 0.2) and incubatedas above for 71 hours.

Example 9 Purification of Baculovirus Expressed Glu-Glu-Tagged zsig32Polypeptides

[0182] Unless otherwise noted, all operations were carried out at 4° C.A mixture of protease inhibitors were added to a 2 liter sample ofconditioned media from C-terminal Glu-Glu (EE) tagged zsig32baculovirus-infected Sf9 cells to final concentrations of 2.5 mMethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co. St. Louis,Mo.), 0.001 mM leupeptin (Boehringer-Mannheim, Indianapolis, Ind.),0.001 mM pepstatin (Boehringer-Mannheim) and 0.4 mM Pefabloc(Boehringer-Mannheim). The sample was centrifuged at 10,000 rpm for 30min at 4° C. in a Beckman JLA-10.5 rotor (Beckman Instruments) in aBeckman Avanti J25I centrifuge (Beckman Instruments) to remove celldebris. To the supernatant fraction-was added a 50.0 ml sample ofanti-EE Sepharose, prepared as described below, and the mixture wasgently agitated on a Wheaton (Millville, N.J.) roller culture apparatusfor 18.0 h at 4° C.

[0183] The mixture was poured into a 5.0×20.0 cm Econo-Column (Bio-RadLaboratories) and the gel was washed with 30 column volumes of phosphatebuffered saline (PBS). The unretained flow-through fraction wasdiscarded. Once the absorbance of the effluent at 280 nM was less than0.05, flow through the column was reduced to zero and the anti-EESepharose gel was washed with 2.0 column volumes of PBS containing 0.2mg/ml of EE peptide (AnaSpec, San Jose, Calif.). The peptide used hasthe sequence Glu-Tyr-Met-Pro-Val-Asp (SEQ ID NO: 19). After 1.0 hour at4° C., flow was resumed and the eluted protein was collected. Thisfraction was referred to as the peptide elution. The anti-EE Sepharosegel was washed with 2.0 column volumes of 0.1 M glycine, pH 2.5, and theglycine wash was collected separately. The pH of the glycine-elutedfraction was adjusted to 7.0 by the addition of a small volume of 10×PBSand stored at 4° C.

[0184] The peptide elution was concentrated to 5.0 ml using a 5,000molecular weight cutoff membrane concentrator (Millipore) according tothe manufacturer's instructions. The concentrated peptide elution wasseparated from free peptide by chromatography on a 1.5×50 cm SephadexG-50 (Pharmacia) column equilibrated in PBS at a flow rate of 1.0 ml/minusing a BioCad Sprint HPLC (PerSeptive BioSystems). Two ml fractionswere collected and the absorbance at 280 nM was monitored. The firstpeak of material absorbing at 280 nM and eluting near the void volume ofthe column was collected. This material represented purified zsig32CEEand was composed of two major bands of apparent molecular weights 19,000and 24,000 on Coomassie Blue-stained SDS-PAGE gels. These bands werepresent in about equimolar amounts. Both bands showed cross-reactivitywith anti-EE antibodies by Western blotting of the purified material.The protein concentration (0.53 mg/ml) of the purified proteins wasdetermined by BCA analysis (Pierce) and the material was aliquoted, andstored at −80° C.

[0185] Preparation of Anti-EE Sepharose

[0186] A 100 ml bed volume of protein G-Sepharose (Pharmacia) was washed3 times with 100 ml of PBS containing 0.02% sodium azide using a 500 mlNalgene 0.45 micron filter unit. The gel was washed with 6.0 volumes of200 mM triethanolamine, pH 8.2 (TEA, Sigma), and an equal volume of EEantibody solution containing 900 mg of antibody was added. After anovernight incubation at 4° C., unbound antibody was removed by washingthe resin with 5 volumes of 200 mM TEA as described above. The resin wasresuspended in 2 volumes of TEA, transferred to a suitable container,and dimethylpimilimidate-2 HCl (Pierce), dissolved in TEA, was added toa final concentration of 36 mg/ml of gel. The gel was rocked at roomtemperature for 45 min and the liquid was removed using the filter unitas described above. Nonspecific sites on the gel were then blocked byincubating for 10 minutes at room temperature with 5 volumes of 20 mMethanolamine in 200 mM TEA. The gel was then washed with 5 volumes ofPBS containing 0.02% sodium azide and stored in this solution at 4° C.

Example 9 Construction of zsig32 Amino Terminal FLAG-Tagged PichiaExpression Vector

[0187] Expression of zsig32 in Pichia methanolica utilizes theexpression system described in co-assigned WIPO publication WO 97/17450.An expression plasmid containing all or part of a polynucleotideencoding zsig32 was constructed via homologous recombination. Theexpression vector was built from pCZR190, which contains the AUG1promoter, followed by the alpha factor prepro (αFpp) leader sequence,followed by an amino-terminal FLAG tag (NF), a blunt-ended Sma Irestriction site for insertion of the gene sequence of interest, atranslational stop codon, followed by the AUG1 terminator, the ADE2selectable marker, and finally the AUG1 3′ untranslated region. Alsoincluded in this vector are the URA3 and CEN-ARS sequences required forselection and replication in S. cerevisiae, and the AmpR and colE1 orisequences required for selection and replication in E. coli. The zsig32sequence inserted into this vector begins at residue 22 (Ala) of thezsig32 amino acid sequence (SEQ ID NO:2).

[0188] Two construct specific linkers were prepared and along withzsig32, were homologously recombined into the yeast expression vectorpCZR190. The N-terminal linker comprises 70 base pairs of the αFppcoding sequence joined to a nucleotide sequence encoding a FLAG tag (SEQID NO:16) followed by 70 base pairs of nucleotide sequence encoding aportion of the amino-terminus from the mature zsig32 sequence. TheC-terminal linker comprises about 70 base pairs of carboxy terminuscoding sequence of the zsig32 joined with 70 base pairs of AUG1terminator sequence.

[0189] The N-terminal linker was synthesized by a PCR reaction. Briefly,to a final reaction volume of 100 μl was added 1 pm each of each linkerZC13735 (SEQ ID NO:29), ZC14291 (SEQ ID NO:30), and 100 pmol of eachprimer ZC13497 (SEQ ID NO:31) and ZC14279 (SEQ ID NO:32), 10 μl of10×PCR buffer (Boehringer Mannheim), 1 μl Two polymerase (BoehringerMannheim), 10 μl of 0.25 mM nucleotide triphosphate mix (Perkin Elmer)and dH₂O. The PCR reaction was incubated at 94° C. for 1.5 minutes,followed by 10 cycles of 30 seconds at 94° C., 1 minute at 50° C. and 1minute at 72° C., concluded with a 10 minute extension at 720. Theresulting 138 bp double stranded, NF-tagged linker is disclosed in SEQID NO:33.

[0190] The C-terminal untagged zsig32 linker was made via a PCR reactionas described using oligonucleotides linkers ZC14346 (SEQ ID NO:34) andZC14218 (SEQ ID NO:35) and primers ZC14278 (SEQ ID NO:36) and ZC13734(SEQ ID NO:37). The resulting 153 bp double stranded, C-terminaluntagged linker is disclosed in SEQ ID NO:38.

[0191] The NF-zsig32 plasmid was made by homologously recombining 100 ngof Sma I digested pCZR202 acceptor vector, the 1 μg of Eco RI-Xho Izsig32 cDNA donor fragment, 1 μg of N-terminal FLAG-tagged zsig32 linker(SEQ ID NO:33) and 1 μg of C-terminal linker (SEQ ID NO:38) into S.cerevisiae. One hundred microliters of competent yeast cells (S.cerevisiae) was combined with 10 μl of each of the fragments and linkersand transferred to a 0.2 cm electroporation cuvette. The yeast/DNAmixture was electropulsed at 0.75 kV (5 kV/cm), ∞ ohms, 25 μF. To thecuvette was added 600 μl of 1.2 M sorbitol and 300 μl aliquots of theyeast/sorbitol mixture was plated onto two URA D plates and incubated at30° C.

[0192] After about 48 hours the Ura⁺ yeast transformants from a singleplate were resuspended in 2.5 ml H₂O and spun briefly to pellet theyeast cells. The cell pellet was resuspended in 1 ml of lysis buffer (2%Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Fivehundred microliters of the lysis mixture was added to an Eppendorf tubecontaining 300 μl acid washed glass beads and 200 μl phenol-chloroform,vortexed for 1 minute intervals two or three times, followed by a 5minute spin in a Eppendorf centrifuge at maximum speed. Three hundredmicroliters of the aqueous phase was transferred to a fresh tube and theDNA precipitated with 600 μl ethanol (EtOH), followed by a 10 minutes at4° C. The DNA pellet was resuspended in 100 μl H₂O.

[0193] Five microliters of the resuspended DNA prep was used totransform 40 μl of electrocompetent E. coli cells (DH10B, Gibco BRL).The cells were electropulsed at 2.0 kV and 400 ohms. Followingelectroporation, 1 ml SOC (2% Bacto™ Tryptone (Difco, Detroit, Mich.),0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 MMMgSO₄, 20 mM glucose) was added and the cells were allowed to recoverfor 1 hour at 37° C. prior to plating 250 μl aliquots on four LB AMPplates (LB broth (Lennox), 1.8% Bacto™ Agar (Difco), 100 mg/LAmpicillin).

[0194] Individual clones harboring the correct expression construct wereidentified by PCR screening. The primers used to amplify the N-taggedzsig32 clone were ZC13479 (SEQ ID NO:31) and ZC13734 (SEQ ID NO:37). Theinsert sequence of positive clones, identified by a 625 bp fragment,were verified by sequence analysis. One such clone was designatedpGMN12-1. Larger scale plasmid DNA was isolated using Qiagen maxi kits(Qiagen) and the DNA was digested with Not I to liberate thePichia-zsig32 expression cassette from the vector backbone. The Not IDNA fragment was then transformed into the Pichia methanolica expressionhost, PMAD16. This was done by mixing 100 μl of prepared competentPMAD16 cells with 10 μg of Not I digested NF-tagged zsig32 fragment andtransferred to a 0.2 cm electroporation cuvette. The yeast/DNA mixturewas electropulsed at 0.75 kV, 25 μF, infinite ohms. To the cuvette wasadded 1 ml of 1×Yeast Nitrogen Base and 500 μl aliquots were plated ontotwo ADE DS (0.056% -Ade -Trp -Thr powder, 0.67% yeast nitrogen basewithout amino acids, 2% D-glucose, 0.5% 200×tryptophan, threoninesolution, and 18.22% D-sorbitol) plates for selection and incubated at30° C. The resulting NF-tagged-zsig32 plasmid was designatedPMAD16::pGMN12-1. Transformants were then picked and screened viaWestern blot for high-level NF-tagged zsig32 expression and subjected tolarge scale fermentation.

[0195] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

1 38 853 base pairs nucleic acid double linear cDNA Coding Sequence168...701 1 GAATTCGGCT CGAGAGGAAG AGCCCCACGG CCAGCTCCTT CCTGTTCCCCTGGCGGCCC 60 TCGCTTCTTC CTTCTGGATG GGGGCCCAGG GGGCCCAGGA GAGTATAAAGGCGATGTG 120 GGGTGCCCGG CACAACCAGA CGCCCAGTCA CAGGCGAGAG CCCTGGG ATG CACCGG 176 Met His Arg 1 CCA GAG GCC ATG CTG CTG CTG CTC ACG CTT GCC CTCCTG GGG GGC CCC 224 Pro Glu Ala Met Leu Leu Leu Leu Thr Leu Ala Leu LeuGly Gly Pro 5 10 15 ACC TGG GCA GGG AAG ATG TAT GGC CCT GGA GGA GGC AAGTAT TTC AGC 272 Thr Trp Ala Gly Lys Met Tyr Gly Pro Gly Gly Gly Lys TyrPhe Ser 20 25 30 35 ACC ACT GAA GAC TAC GAC CAT GAA ATC ACA GGG CTG CGGGTG TCT GTA 320 Thr Thr Glu Asp Tyr Asp His Glu Ile Thr Gly Leu Arg ValSer Val 40 45 50 GGT CTT CTC CTG GTG AAA AGT GTC CAG GTG AAA CTT GGA GACTCC TGG 368 Gly Leu Leu Leu Val Lys Ser Val Gln Val Lys Leu Gly Asp SerTrp 55 60 65 GAC GTG AAA CTG GGA GCC TTA GGT GGG AAT ACC CAG GAA GTC ACCCTG 416 Asp Val Lys Leu Gly Ala Leu Gly Gly Asn Thr Gln Glu Val Thr Leu70 75 80 CAG CCA GGC GAA TAC ATC ACA AAA GTC TTT GTC GCC TTC CAA GCT TTC464 Gln Pro Gly Glu Tyr Ile Thr Lys Val Phe Val Ala Phe Gln Ala Phe 8590 95 CTC CGG GGT ATG GTC ATG TAC ACC AGC AAG GAC CGC TAT TTC TAT TTT512 Leu Arg Gly Met Val Met Tyr Thr Ser Lys Asp Arg Tyr Phe Tyr Phe 100105 110 115 GGG AAG CTT GAT GGC CAG ATC TCC TCT GCC TAC CCC AGC CAA GAGGGG 560 Gly Lys Leu Asp Gly Gln Ile Ser Ser Ala Tyr Pro Ser Gln Glu Gly120 125 130 CAG GTG CTG GTG GGC ATC TAT GGC CAG TAT CAA CTC CTT GGC ATCAAG 608 Gln Val Leu Val Gly Ile Tyr Gly Gln Tyr Gln Leu Leu Gly Ile Lys135 140 145 AGC ATT GGC TTT GAA TGG AAT TAT CCA CTA GAG GAG CCG ACC ACTGAG 656 Ser Ile Gly Phe Glu Trp Asn Tyr Pro Leu Glu Glu Pro Thr Thr Glu150 155 160 CCA CCA GTT AAT CTC ACA TAC TCA GCA AAC TCA CCC GTG GGT CGCTAG 706 Pro Pro Val Asn Leu Thr Tyr Ser Ala Asn Ser Pro Val Gly Arg 165170 175 TGGGGTATGG GGCCATCCGA GCTGAGGCCA TCTGTGTGGT GGTGGCTGAT GGTACTGG766 TAACTGAGTC GGGACGCTGA ATCTGAATCC ACCAATAAAT AAAGCTTCTG CAGAATCA 826GAAAAAAAAA AAAAAAAGGG CGGCCGC 853 178 amino acids amino acid singlelinear protein internal 2 Met His Arg Pro Glu Ala Met Leu Leu Leu LeuThr Leu Ala Leu Leu 1 5 10 15 Gly Gly Pro Thr Trp Ala Gly Lys Met TyrGly Pro Gly Gly Gly Lys 20 25 30 Tyr Phe Ser Thr Thr Glu Asp Tyr Asp HisGlu Ile Thr Gly Leu Arg 35 40 45 Val Ser Val Gly Leu Leu Leu Val Lys SerVal Gln Val Lys Leu Gly 50 55 60 Asp Ser Trp Asp Val Lys Leu Gly Ala LeuGly Gly Asn Thr Gln Glu 65 70 75 80 Val Thr Leu Gln Pro Gly Glu Tyr IleThr Lys Val Phe Val Ala Phe 85 90 95 Gln Ala Phe Leu Arg Gly Met Val MetTyr Thr Ser Lys Asp Arg Tyr 100 105 110 Phe Tyr Phe Gly Lys Leu Asp GlyGln Ile Ser Ser Ala Tyr Pro Ser 115 120 125 Gln Glu Gly Gln Val Leu ValGly Ile Tyr Gly Gln Tyr Gln Leu Leu 130 135 140 Gly Ile Lys Ser Ile GlyPhe Glu Trp Asn Tyr Pro Leu Glu Glu Pro 145 150 155 160 Thr Thr Glu ProPro Val Asn Leu Thr Tyr Ser Ala Asn Ser Pro Val 165 170 175 Gly Arg 199amino acids amino acid single linear peptide 3 Met Leu Leu Leu Leu ThrLeu Ala Phe Leu Ala Ser Pro Thr Cys Arg 1 5 10 15 Ala Gln Asn Val LeuGly Asn Ala Ala Gly Lys Tyr Phe Tyr Val Gln 20 25 30 Gly Glu Asp Gln GlyGln Leu Lys Gly Met Arg Ile Phe Leu Ser Val 35 40 45 Phe Lys Phe Ile LysGly Phe Gln Leu Gln Phe Gly Ser Asn Trp Thr 50 55 60 Asp Val Tyr Gly ThrArg Ser Asp Asn Phe Ile Asp Phe Leu Leu Glu 65 70 75 80 Asp Gly Glu HisVal Ile Lys Val Glu Gly Ser Ala Val Ile Cys Leu 85 90 95 hr Ser Leu ThrPhe Thr Thr Asn Lys Gly Arg Val Ala Thr Phe Gly 100 105 110 Val Arg ArgGly Arg Tyr Phe Ser Asp Thr Gly Gly Ser Asp Lys His 115 120 125 Leu ValThr Val Asn Gly Met His Ala Pro Gly Leu Cys Val Arg Gly 130 135 140 IleGly Phe Lys Trp Gly Asn Ile Asn Ala Asn Gly Asn Asp His Tyr 145 150 155160 Asn Asn Lys Glu Asp Lys Ala Asp Asn Lys Asp Ala Asp Asn Lys Asp 165170 175 Ala Asp Asn Lys Asp Asp Gly Asp Glu Asp Asp Asp Gly Asn Asp Asp180 185 190 Asp Asp Gln Lys Asp Glu Ser 195 166 amino acids amino acidsingle linear peptide 4 Met Leu Pro Gln Leu Glu Ala Met Leu Pro Leu LeuIle Leu Ala Phe 1 5 10 15 Leu Gly Thr Pro Ala Val Leu Thr Gln Ser ArgTyr His Gly Ser Glu 20 25 30 Thr Gly Lys His Phe Cys Ile Val Ala Pro GluGly Glu Pro Val Thr 35 40 45 Gly Ile Trp Ala Ser Leu Lys Asn Asn Ile LeuSer Ser Ile Arg Leu 50 55 60 Lys Phe Gly Asn Asn Trp Ser Gln Glu Tyr GlySer Ser Gly Arg Ala 65 70 75 80 Glu Ile Glu Val Lys Leu Asn Pro Asp GluThr Val Leu Gly Phe Ser 85 90 95 Gly Ser Phe Tyr Ile Phe Met His Gln IleIle Ile Thr Thr Ser Gln 100 105 110 Pro Arg Glu Leu Ile Ile Gly Pro LeuThr Gly Arg Tyr Val Tyr Thr 115 120 125 Ser Tyr Pro Glu Asn Pro Asn HisVal Phe Arg Gly Ile Cys Gly Tyr 130 135 140 Tyr Val Thr Gly Gly Leu LysGly Met Arg Tyr Leu Trp Gly Asn Val 145 150 155 160 Asn Gly Thr Cys ThrGlu 165 170 amino acids amino acid single linear peptide 5 Met Phe GlnLeu Glu Ala Met Leu Pro Leu Leu Ile Leu Ala Phe Leu 1 5 10 15 Gly ThrPro Thr Val Leu Thr Gln Asp Tyr His Gly Pro Glu Val Gly 20 25 30 Lys HisSer Cys Thr Ser Ala Pro Glu Gly Lys Asn Ile Thr Ser Ile 35 40 45 Arg ValPhe Leu Gln Gly Arg Ser Ile Val Gly Ile Gln Phe Asn Tyr 50 55 60 Asn AsnGlu Asp Gly Gln Val Tyr Gly Ser Thr Ala Gly Lys Val Met 65 70 75 80 ValAla Arg Leu Asn Asn Glu Glu Ser Ile Ile Ala Ala Glu Gly Thr 85 90 95 TyrSer Pro Ser Ala Leu Thr Gln Ile Ile Phe Thr Thr Asn Gln Pro 100 105 110Arg Gln Leu Met Val Gly Tyr Tyr Val Gly Ser Ser Glu Tyr Ser Ser 115 120125 Phe Pro Asp Asp Pro Ser His Val Leu Lys Gly Ala Cys Val Ser Trp 130135 140 Arg Ala Gly Gly Ile Lys Ser Ile Leu Phe Leu Trp Gly Thr Glu Asn145 150 155 160 Ser Ser Cys Val Lys Tyr Gly His Ser Gly 165 170 15 aminoacids amino acid single linear peptide 6 Leu Leu Thr Leu Ala Leu Leu GlyGly Pro Thr Trp Ala Gly Lys 1 5 10 15 40 base pairs nucleic acid singlelinear Other ZC12493 7 GGTGCTGAAA TACTTGCCTC CTCCAGGGCC ATACATCTTC 40 20base pairs nucleic acid single linear Other ZC694 8 TAATACGACTCACTATAGGG 20 19 base pairs nucleic acid single linear Other ZC695 9GATTTAGGTG ACACTATAG 19 20 base pairs nucleic acid single linear OtherZC13183 10 ATAGAAATAG CGGTCCTTGC 20 20 base pairs nucleic acid singlelinear Other ZC13187 11 GCAGCCAGGC GAATACATCA 20 7 amino acids aminoacid single linear peptide 12 Glu Glu Tyr Met Pro Met Glu 1 5 38 basepairs nucleic acid single linear cDNA ZC13404 13 GGCGAGAGGA TCCGCATGCACCGGCCAGAG GCCATGCT 38 34 base pairs nucleic acid single linear cDNAZC13409 14 CCGGTACCTA GCGACCCACG GGTGAGTTTG CTGA 34 55 base pairsnucleic acid single linear cDNA ZC13407 15 CCGGTACCTA TTCCATCGGCATGTATTCTT CGCGACCCAC GGGTGAGTTT GCTGA 55 8 amino acids amino acidsingle linear peptide 16 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 18 basepairs nucleic acid single linear cDNA ZC976 17 CGTTGTAAAA CGACGGCC 18 17base pairs nucleic acid single linear cDNA ZC447 18 TAACAATTTC ACACAGG17 6 amino acids amino acid single linear peptide 19 Glu Tyr Met Pro ValAsp 1 5 534 base pairs nucleic acid single linear Other 20 ATGCAYMGNCCNGARGCNAT GYTNYTNYTN YTNACNYTNG CNYTNYTNGG NGGNCCNAC 60 TGGGCNGGNAARATGTAYGG NCCNGGNGGN GGNAARTAYT TYWSNACNAC NGARGAYT 120 GAYCAYGARATHACNGGNYT NMGNGTNWSN GTNGGNYTNY TNYTNGTNAA RWSNGTNC 180 GTNAARYTNGGNGAYWSNTG GGAYGTNAAR YTNGGNGCNY TNGGNGGNAA YACNCARG 240 GTNACNYTNCARCCNGGNGA RTAYATHACN AARGTNTTYG TNGCNTTYCA RGCNTTYY 300 MGNGGNATGGTNATGTAYAC NWSNAARGAY MGNTAYTTYT AYTTYGGNAA RYTNGAYG 360 CARATHWSNWSNGCNTAYCC NWSNCARGAR GGNCARGTNY TNGTNGGNAT HTAYGGNC 420 TAYCARYTNYTNGGNATHAA RWSNATHGGN TTYGARTGGA AYTAYCCNYT NGARGARC 480 ACNACNGARCCNCCNGTNAA YYTNACNTAY WSNGCNAAYW SNCCNGTNGG NMGN 534 18 base pairsnucleic acid single linear cDNA ZC13703 21 CCTGGGACGT GAAACTGG 18 18base pairs nucleic acid single linear cDNA ZC13704 22 TGGAAGGCGACAAAGACT 18 25 base pairs nucleic acid single linear cDNA ZC13465 23GCGGGATCC GCGACCCACG GGTGA 25 25 base pairs nucleic acid single linearcDNA ZC13447 24 GCGCGAATTC ATGCACCGGC CAGAG 25 25 base pairs nucleicacid single linear cDNA ZC13448 25 CGCGCTCGAG CTAGCGACCC ACGGG 25 25base pairs nucleic acid single linear cDNA ZC13449 26 GCGCGGATCCGGGAAGATGT ATGGC 25 21 base pairs nucleic acid single linear cDNA ZC658327 GTCCAACGAC TATAAAGAGG G 21 21 base pairs nucleic acid single linearcDNA ZC5020 28 CACTGGAGTG GCAACTTCCA G 21 51 base pairs nucleic acidsingle linear cDNA ZC13735 29 GGTGTAAGCT TGGACAAGAG AGATTACAAGGACGATGATG ACAAGGGTGG T 51 66 base pairs nucleic acid single linear cDNAZC14291 30 TGGTGCTGAA ATACTTGCCT CCTCCAGGGC CATACATCTT CCCACCACCCTTGTCATCA 60 CGTCCT 66 44 base pairs nucleic acid single linear cDNAZC14297 31 AGCATTGCTG CTAAAGAAGA AGGTGTAAGC TTGGACAAGA GAGA 44 50 basepairs nucleic acid single linear cDNA ZC14279 32 CTGTGATTTC ATGGTCGTAGTCTTCAGTGG TGCTGAAATA CTTGCCTCCT 50 139 base pairs nucleic acid singlelinear cDNA 33 AGCATTGCTG CTAAAGAAGA AGGTGTAAGC TTGGACAAGA GAGATTACAAGGACGATGA 60 GACAAGGGTG GTGGGAAGAT GTATGGCCCT GGAGGAGGCA AGTATTTCAGCACCACTG 120 GACTACGACC ATGAAATCA 139 68 base pairs nucleic acid singlelinear cDNA ZC14346 34 CAGTTAATCT CACATACTCA GCAAACTCAC CCGTGGGTCGCTAGGAATTC TAGTATTCT 60 GGGCTGCC 68 60 base pairs nucleic acid singlelinear cDNA ZC14218 35 TGGCAAACTC TCAAAAATTA TAAAAATATC CAAACAGGCAGCCCTAGAAT ACTAGAATT 60 51 base pairs nucleic acid single linear cDNAZC14278 36 ACTAGAGGAG CCGACCACTG AGCCACCAGT TAATCTCACA TACTCAGCAA A 5152 base pairs nucleic acid single linear cDNA ZC13734 37 ATCATAGAAGAGAAAAACAT TAGTTGGCAA ACTCTCAAAA ATTATAAAAA TA 52 154 base pairs nucleicacid single linear cDNA 38 ACTAGAGGAG CCGACCACTG AGCCACCAGT TAATCTCACATACTCAGCAA ACTCACCCG 60 GGGTCGCTAG GAATTCTAGT ATTCTAGGGC TGCCTGTTTGGATATTTTTA TAATTTTT 120 GAGTTTGCCA ACTAATGTTT TTCTCTTCTA TGAT 154

What is claimed is:
 1. An isolated polypeptide comprising a sequence ofamino acid residues that is at least 80% identical in amino acidsequence to residues 23-178 of SEQ ID NO:2.
 2. An isolated polypeptideaccording to claim 1, wherein said polypeptide is at least 90% identicalin amino acid sequence to residues 23-178 of SEQ ID NO:2.
 3. An isolatedpolypeptide according to claim 1, wherein said polypeptide comprisesresidues 7-178 of SEQ ID NO:2.
 4. An isolated polypeptide according toclaim 1, wherein said polypeptide comprises residues 1-178 of SEQ IDNO:2.
 5. An isolated polypeptide according to claim 1, wherein saidpolypeptide is at least 1 kb in length.
 6. An isolated polypeptideaccording to claim 1, covalently linked to a moiety selected from thegroup consisting of affinity tags, toxins, radionucleotides, enzymes andfluorophores.
 7. An isolated polypeptide according to claim 7, whereinsaid moiety is an affinity tag selected from the group consisting ofpolyhistidine, FLAG, Glu-Glu, glutathione S transferase and animmunoglobulin heavy chain constant region.
 8. An isolated polypeptideaccording to claim 7 further comprising a proteolytic cleavage sitebetween said sequence of amino acid residues and said affinity tag. 9.An expression vector comprising the following operably linked elements:a transcription promoter; a DNA segment encoding a polypeptidecomprising a sequence of amino acid residues that is at least 80%identical in amino acid sequence to residues 23-178 of SEQ ID NO:2; anda transcriptional terminator.
 10. An expression vector according toclaim 9, wherein said DNA segment encodes a polypeptide is at least 90%identical in amino acid sequence to residues 23-178 of SEQ ID NO:2. 11.An expression vector according to claim 9, wherein said polypeptideencoded by said DNA segment comprises residues 7-178 of SEQ ID NO:2. 12.An expression vector according to claim 9, wherein said polypeptideencoded by said DNA segment comprises residues 1-178 of SEQ ID NO:2. 13.An expression vector according to claim 9, wherein said DNA segmentencodes a polypeptide covalently linked to an affinity tag selected fromthe group consisting of polyhistidine, FLAG, Glu-Glu, glutathione Stransferase and an immunoglobulin heavy chain constant region.
 14. Anexpression vector according to claim 9 wherein said DNA further encodesa secretory signal sequence operably linked to said polypeptide.
 15. Anexpression vector according the claim 9, wherein said secretory signalsequence encodes residues 7-22 of SEQ ID NO:2.
 16. An expression vectoraccording the claim 9, wherein said secretory signal sequence encodesresidues 1-22 of SEQ ID NO:2.
 17. A cultured cell into which has beenintroduced an expression vector comprising the following operably linkedelements: a transcription promoter; a DNA segment encoding a polypeptidecomprising a sequence of amino acid residues that is at least 80%identical in amino acid sequence to residues 23-178 of SEQ ID NO:2; anda transcriptional terminator, wherein said cell expresses saidpolypeptide encoded by said DNA segment.
 18. A method of producing aprotein comprising: culturing a cell into which has been introduced anexpression vector comprising the following operably linked elements: atranscription promoter; a DNA segment encoding a polypeptide comprisinga sequence of amino acid residues that is at least 80% identical inamino acid sequence to residues 23-178 of SEQ ID NO:2; and atranscriptional terminator, whereby said cell expresses said proteinencoded by said DNA segment; and recovering said expressed protein. 19.A pharmaceutical composition comprising a polypeptide that is at least80% identical in amino acid sequence to residues 23-178 of SEQ ID NO:2,in combination with a pharmaceutically acceptable vehicle.
 20. Anantibody that specifically binds to an epitope of a polypeptide of SEQID NO:2.
 21. A binding protein that specifically binds to an epitope ofa polypeptide of SEQ ID NO:2.
 22. An isolated polynucleotide encoding apolypeptide comprising a sequence of amino acid residues that is atleast 80% identical in amino acid sequence to residues 23-178 of SEQ IDNO:2.
 23. An isolated polynucleotide according to claim 22, wherein saidpolypeptide is at least 90% identical in amino acid sequence to residues23-178 of SEQ ID NO:2.
 24. An isolated polynucleotide according to claim22, wherein said polypeptide comprises residues 7-178 of SEQ ID NO:2.25. An isolated polynucleotide according to claim 22, wherein saidpolypeptide comprises residues 1-178 of SEQ ID NO:2.
 26. An isolatedpolynucleotide according to claim 22, wherein said polypeptide isapproximately 1 kb in length.
 27. An isolated polynucleotide accordingto claim 22, wherein said polynucleotide is from 471 to 853 nucleotidesin length.
 28. An isolated polynucleotide according to claim 22comprising nucleotide 1 to nucleotide 534 of SEQ ID NO:20.
 29. Anisolated polynucleotide according to claim 22, wherein saidpolynucleotide is DNA.
 30. An isolated polynucleotide selected from thegroup consisting of, a) a sequence of nucleotides from nucleotide 168 tonucleotide 704 of SEQ ID NO:1; b) a sequence of nucleotides fromnucleotide 186 to nucleotide 704 of SEQ ID NO:2; c) a sequence ofnucleotides from nucleotide 234 to nucleotide 704 of SEQ ID NO:2; d) asequence of nucleotides from nucleotide 246 to nucleotide 704 of SEQ IDNO:2; e) orthologs of a), b), c) or d); f) degenerate sequences of a),b), c), d) or e); and g) nucleotide sequences complementary to a) b) c),d), e) or f).
 31. An oligonucleotide probe or primer comprising 14contiguous nucleotides of a polynucleotide of SEQ ID NO:20 or a sequencecomplementary to SEQ ID NO:20.
 32. A method for detecting a geneticabnormality in a patient, comprising: obtaining a genetic sample from apatient; incubating the genetic sample with a polynucleotide comprisingat least 14 contiguous nucleotides of SEQ ID NO:1 or the complement ofSEQ ID NO:1, under conditions wherein said polynucleotide will hybridizeto complementary polynucleotide sequence, to produce a first reactionproduct; comparing said first reaction product to a control reactionproduct, wherein a difference between said first reaction product andsaid control reaction product is indicative of a genetic abnormality inthe patient.
 33. A DNA construct encoding a polypeptide fusion, saidfusion comprising a secretory signal sequence selected from the groupconsisting of: (a) amino acid residues 1-22 of SEQ ID NO:2; and (b)amino acid residues 7-22 of SEQ ID NO:2; wherein said secretory signalsequence is operably linked to an additional polypeptide.